Chd Candidate Gene Emc1 Loss Of Function Drives Neural Crest And Transmembrane Protein Defects

Recent advances in genetic sequencing technologies have enabled identification of numerous congenital heart disease (CHD) candidate genes. One such gene, endoplasmic reticulum transmembrane complex 1 (EMC1), has been implicated in proper transmembrane protein topogenesis and associated with varied patient phenotypes. Using a western clawed frog (Xenopus tropicalis—X. tropicalis) animal model, we recapitulate a cardiac phenotype with EMC1 loss of function and identify additional pigment and craniofacial phenotypes consistent with a neural crest cell (NCC) pathology, which we corroborate at the molecular level. We subsequently demonstrate that EMC1 loss of function causes abnormal WNT signaling, as manifested in abnormal β-Catenin levels and localization, and establish a plausible mechanism for abnormal NCC development in which EMC1 loss of function affects the transmembrane WNT signal transducers FZD2 and FZD7. We additionally show that EMC1 loss of function affects expression and/or localization of the transmembrane proteins rhodopsin and the nicotinic acetylcholine receptor (nAChR) and confirm that EMC1 loss of function triggers an un-folded protein response, likely mediated by accumulation of mis-localized transmembrane proteins. Altogether, our work demonstrates a complex and yet elegantly plausible model for CHD pathogenesis in patients with EMC1 mutations, including the unexpected twist of NCC pathology and a unifying theory for other patient phenotypes

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD⁺ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.National Institutes of Health (U.S.) (Grant R01- ES022872)Ellison Medical Foundation (Award AG-SS-3046-12

mAagTg CGN sensitivity to MMS is not dependent on NAD+, pyruvate, caspases, Rip1K, calcium fluxes, or MPT.

<p>(A) NAD⁺ pre-treatment did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 3. (B) Pyruvate did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 1. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS and MMS + Veliparib. (C) Primary <i>mAagTg</i> CGN sensitivity to MMS is not dependent on caspase activation, Rip1 kinase activity, calcium fluxes, or mitochondrial permeability transition (MPT). Inhibitors used include zVad-fmk (zVad), n = 6, Necrostatin-1 (Nec-1), n = 3, BAPTA-AM, n = 3, and cyclosporin A (CsA), n = 3. Errors bars denote standard error from the mean. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS to MMS+Veliparib.</p

No loss in mitochondrial permeability or translocation of AIF in neurons after MMS treatment.

<p>(A) There is no loss of mitochondrial permeability in <i>Aag</i>^-/-, WT, or <i>mAagTg</i> neurons 1–7 hours after MMS treatment. Errors bars denote standard deviation from the mean. <i>Aag</i>^-/- n = 2, WT n = 3, <i>mAagTg</i> n = 2. ** p<0.01 using Student’s standard two-tailed T-test comparing FCCP to control. * p<0.05 using Student’s standard one-tailed T-test comparing FCCP to control. (B) WT CGNs treated with MMS do not show evidence of AIF (red) translocation from the mitochondria (green; CoxIV) to the nucleus (blue; Hoechst).</p

CGN sensitivity to MMS is dependent on Aag and involves activation of Parp.

<p>(A) Overview of CGN toxicity assay based on high content imaging. (B) Sensitivity to MMS treatment <i>ex vivo</i> is dependent on Aag. Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test comparing a single dose to WT. (C) PAR formation (green) was visualized in <i>Aag</i>^-/-, WT, and <i>mAagTg</i> neurons 0, 15, 30, or 60 minutes after the addition of MMS (1 mM) by immunocytochemical staining. (D) Fold changes in nuclear PAR staining was quantified and reflects qualitative trends in (C). Errors bars denote standard error from the mean. <i>Aag</i>^-/-: n = 4, WT: n = 3, <i>mAagTg</i>: n = 2. **p<0.01 using Student’s standard two-tailed T-test comparing nuclear fluorescence at a particular timepoint compared to untreated cells. (E) Representative images of PAR formation in <i>mAagTg</i> neurons either left untreated, or 15 minutes after MMS treatment (1 mM) with or without the addition of Veliparib (5 μM). (F) Quantification of changes in nuclear PAR formation in neurons with or without Veliparib pretreatment. Errors bars denote standard error from the mean. <i>Aag</i>^-/-: n = 4, WT: n = 3, <i>mAagTg</i>: n = 2. * p<0.05 using Student’s standard two-tailed T-test comparing nuclear fluorescence with or without Veliparib at a particular time post MMS treatment.</p

Isolation and characterization of <i>Aag</i>^-/- cerebellar granule neurons.

<p>(A) Neurons were isolated from mouse cerebella at post-natal day 5–8. Representative immunocytochemical image depicts neurons (Map2, green) and astrocytes (GFAP, red). Nuclei are shown in blue. (B) Map2+ neurons were quantified from images from 10 pups isolated on different days. Error bars represent standard deviation from the mean. (C) <i>Aag</i> transcripts were counted per cell and are plotted by genotype, where each data point represents one cell. Errors bars denote standard deviation from the mean, n ≥ 50 cells, *** p<0.001 using Student’s standard two-tailed T-test. (D) Cartoon representation of the host-cell reactivation method to measure Aag glycosylase activity in cells. (E) Glycosylase activity over time by cellular fluorescent output from host cell reactivation assay. Solid lines denote mean while dashed lines indicate standard deviation from the mean. (F) WT, <i>Aag</i>^-/- and <i>mAagTg</i> neurons have significantly different Aag glycosylase activity at 24 hours post-transfection. Errors bars denote standard deviation from the mean. WT n = 3, <i>mAagTg</i> n = 2, <i>Aag</i>^-/- n = 2. ** p<0.01 using Student’s standard two-tailed T-test). (G) Fold changes between WT, <i>Aag</i>^-/- and <i>mAagTg</i> Aag expression and glycosylase activity.</p

Parp inhibition rescues CGN sensitivity to MMS.

<p><i>Aag</i>^-/-, WT, and <i>mAagTg</i> neurons are rescued from MMS toxicity (1 mM) after pretreatment with Parp inhibitor Veliparib (A) or Olaparib (B). Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test compared to neurons of the same genotype treated with 1 mM MMS (control). (C) Parp inhibitor Veliparib rescues cell sensitivity at all doses of MMS in WT and <i>mAagTg</i> neurons. Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test comparing sensitivity with or without Veliparib for a genotype at a particular dose of MMS. (D) Expression of BER genes in CGNs. Errors bars denote standard deviation from the mean, <i>Aag</i>^-/- n = 4, WT n = 5, <i>mAagTg</i> n = 1. (E) Aag expression was assessed either before, immediately after, or an hour after MMS treatment using <i>Aag</i> mRNA FISH in WT CGNs. Errors bars denote standard deviation from the mean.</p