NMNAT2 is downregulated in glaucomatous RGCs, and RGC-specific gene therapy rescues neurodegeneration and visual function

Molecular therapy : the journal of the American Society of Gene Therapy

2022 Jan 31

Fang, F;Zhuang, P;Feng, X;Liu, P;Liu, D;Huang, H;Li, L;Chen, W;Liu, L;Sun, Y;Jiang, H;Ye, J;Hu, Y;
PMID: 35114390 | DOI: 10.1016/j.ymthe.2022.01.035

The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse γ-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline, and that modulating RGC-intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

Spatial expression of IKK-alpha is associated with a differential mutational landscape and survival in primary colorectal cancer

British journal of cancer

2022 Feb 16

Patel, M;Pennel, KAF;Quinn, JA;Hood, H;Chang, DK;Biankin, AV;Rebus, S;Roseweir, AK;Park, JH;Horgan, PG;McMillan, DC;Edwards, J;
PMID: 35173303 | DOI: 10.1038/s41416-022-01729-2

To understand the relationship between key non-canonical NF-κB kinase IKK-alpha(α), tumour mutational profile and survival in primary colorectal cancer.Immunohistochemical expression of IKKα was assessed in a cohort of 1030 patients who had undergone surgery for colorectal cancer using immunohistochemistry. Mutational tumour profile was examined using a customised gene panel. Immunofluorescence was used to identify the cellular location of punctate IKKα expression.Two patterns of IKKα expression were observed; firstly, in the tumour cell cytoplasm and secondly as discrete 'punctate' areas in a juxtanuclear position. Although cytoplasmic expression of IKKα was not associated with survival, high 'punctate' IKKα expression was associated with significantly reduced cancer-specific survival on multivariate analysis. High punctate expression of IKKα was associated with mutations in KRAS and PDGFRA. Dual immunofluorescence suggested punctate IKKα expression was co-located with the Golgi apparatus.These results suggest the spatial expression of IKKα is a potential biomarker in colorectal cancer. This is associated with a differential mutational profile highlighting possible distinct signalling roles for IKKα in the context of colorectal cancer as well as potential implications for future treatment strategies using IKKα inhibitors.

Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease

Brain

2021 Sep 30

Steinkellner, T;Conrad, WS;Kovacs, I;Rissman, RA;
| DOI: 10.1093/brain/awab373

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson’s disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving DA neurons from postmortem Parkinson’s disease subjects. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson’s disease.

Scientific Evidence Supporting Coronavirus Disease 2019 (COVID-19) Vaccine Efficacy and Safety in People Planning to Conceive or Who Are Pregnant or Lactating

Obstetrics and gynecology

2021 Nov 02

Girardi, G;Bremer, AA;
PMID: 34727554 | DOI: 10.1097/AOG.0000000000004636

Three coronavirus disease 2019 (COVID-19) vaccines have been authorized for use in the United States; specifically, the Pfizer-BioNTech, Moderna, and Johnson & Johnson-Janssen COVID-19 vaccines were granted emergency use authorization by the U.S. Food and Drug Administration in late 2020 and early 2021. Vaccination coverage and intent among adults are lowest among those aged 18-39 years and among females in particular. In females of reproductive age, enthusiasm for receiving a COVID-19 vaccine may be negatively affected by claims currently circulating widely on diverse social media platforms regarding the vaccines adversely affecting fertility and pregnancy. Yet it is important to note that these claims are anecdotal in nature and not supported by the available scientific evidence. It is also imperative that the effects of COVID-19 vaccine on reproductive health are clarified. Herein, we discuss the existing scientific data supporting COVID-19 vaccine safety and efficacy in people who are planning to conceive or who are pregnant or lactating and highlight the importance of COVID-19 vaccination in females of reproductive age.

Quantitative Off-Target Detection of Epstein-Barr Virus-Derived DNA in Routine Molecular Profiling of Hematopoietic Neoplasms by Panel-Based Hybrid-Capture Next-Generation Sequencing

The Journal of molecular diagnostics : JMD

2021 Nov 18

Petrova-Drus, K;Quesada, AE;Bowman, AS;Ptashkin, R;Yao, J;Arcila, ME;Ho, C;Moung, C;Regalado, J;Benayed, R;Benhamida, JK;Galera, PK;Dogan, A;Vanderbilt, C;
PMID: 34801704 | DOI: 10.1016/j.jmoldx.2021.10.009

Epstein-Barr virus (EBV) is associated with hematologic and solid tumors. In this study, we utilized a hybridization capture-based next-generation sequencing (NGS) platform that targets 400 genes associated with hematological malignancies to detect and quantify nontargeted viral-derived EBV reads that aligned to the EBV reference contig (NC_007605). We evaluated 5234 samples from 3636 unique patients with hematological neoplasms and found that 100 samples (1.9%) in 93 unique patients had ≥6 EBV reads (range, 6 to 32,325; mean, 827.5; median, 54). Most (n = 73, 73%) represented known EBV-associated conditions, and the most common was post-transplant lymphoproliferative disorders (n = 21, 29%). Documented EBV viremia accounted for a moderate quantity of EBV reads in 4 of 27 samples corresponding to conditions not known to be EBV associated, whereas suspected viremia or low-level activation was likely the etiology in the remaining 23 samples. A good correlation (Spearman r = 0.8; 95% CI, 0.74-0.85) was found between EBV reads by NGS and systematic semiquantitative EBER in situ hybridization assessment in 162 available samples, particularly at higher level of EBV involvement. An optimal threshold for significant morphologic EBV involvement was found to be ≥10 reads by the receiver operating characteristic analysis (area under the curve, 0.990; 95% CI, 0.9974%-1.000%). Thus, in addition to mutational analysis, hybrid-capture-based NGS panels can be used to detect and quantitate off-target EBV-derived viral DNA, which correlates well with morphology.

Molecular mechanisms in IL-1β-mediated decorin production by decidual cells

Molecular human reproduction

2021 Nov 27

Halari, CD;Renaud, SJ;Lala, PK;
PMID: 34915564 | DOI: 10.1093/molehr/gaab068

Decorin, a small leucine-rich proteoglycan produced by decidual cells restrains trophoblast differentiation, migration and invasiveness of extra-villous trophoblast cells. Decidual overproduction of decorin is associated with preeclampsia, and elevated decorin levels in maternal plasma are a predictive biomarker of preeclampsia. Furthermore, decorin plays an autocrine role in maturation of human endometrial stromal cells into decidual cells. Thus, a balanced decorin production by the decidua is critical for healthy pregnancy. However, the molecular mechanisms regulating decorin production by the decidua are unclear. Interleukin-1 beta is an inflammation-associated multi-functional cytokine, and is reported to induce decidualization in primates. Hence, the present study was designed: (i) to test if exogenous Interleukin-1 beta stimulated decorin production by human endometrial stromal cells; and if so, (ii) to identify the cellular source of Interleukin-1 beta in first trimester decidual tissue; (iii) to identify the downstream molecular partners in Interleukin-1 beta mediated decorin production by human endometrial stromal cells. Results revealed that (i) amongst multiple pro-inflammatory cytokines tested, Interleukin-1 beta alone stimulated decorin production by these cells; (ii) both macrophages and decidual cells in first trimester decidua produced Interleukin-1 beta; (iii) Interleukin-1 beta mediated decorin production was dependent on Interleukin-1 receptor activation, followed by activation and nuclear translocation of nuclear factor kappa B and its binding to the decorin promoter. These results reveal that Interleukin-1 beta plays a novel role in inducing decorin production by human endometrial stromal cells by activating nuclear factor kappa B.

Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry

Nature communications

2021 Oct 08

van Maldegem, F;Valand, K;Cole, M;Patel, H;Angelova, M;Rana, S;Colliver, E;Enfield, K;Bah, N;Kelly, G;Tsang, VSK;Mugarza, E;Moore, C;Hobson, P;Levi, D;Molina-Arcas, M;Swanton, C;Downward, J;
PMID: 34625563 | DOI: 10.1038/s41467-021-26214-x

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.

Naked (N) mutant mice carry a nonsense mutation in the homeobox of Hoxc13

Experimental dermatology

2021 Oct 16

Perez, CJ;Mecklenburg, L;Fernandez, A;Cantero, M;de Souza, TA;Lin, K;Dent, SYR;Montoliu, L;Awgulewitsch, A;Benavides, F;
PMID: 34657330 | DOI: 10.1111/exd.14469

Loss of function mutations in HOXC13 have been associated with Ectodermal Dysplasia-9, Hair/Nail Type (ECTD9) in consanguineous families, characterized by sparse to complete absence of hair and nail dystrophy. Here we characterize the spontaneous mouse mutation Naked (N) as a terminal truncation in the Hoxc13 (homeobox C13) gene. Similar to previous reports for homozygous Hoxc13 knock-out (KO) mice, homozygous N/N mice exhibit generalized alopecia with abnormal nails and a short lifespan. However, in contrast to Hoxc13 heterozygous KO mice, N/+ mice show generalized or partial alopecia, associated with loss of hair fibres, along with normal lifespan and fertility. Our data point to a lack of nonsense-mediated Hoxc13 transcript decay and the presence of the truncated mutant protein in N/N and N/+ hair follicles, thus suggesting a dominant-negative mutation. To our knowledge, this is the first report of a semi-dominant and potentially dominant-negative mutation affecting Hoxc13/HOXC13. Furthermore, recreating the N mutant allele in mice using CRISPR/Cas9-mediated genome editing resulted in the same spectrum of deficiencies as those associated with the spontaneous Naked mutation, thus confirming that N is indeed a Hoxc13 mutant allele. Considering the low viability of the Hoxc13 KO mice, the Naked mutation provides an attractive new model for studying ECTD9 disease mechanisms.

Reproductive Deficits Induced by Prenatal Anti-Mullerian Hormone Exposure Require Androgen Receptor in Kisspeptin Cells

Endocrinology

2021 Sep 16

Ho, EV;Shi, C;Cassin, J;He, MY;Nguyen, RD;Ryan, GE;Tonsfeldt, KJ;Mellon, PL;
PMID: 34529765 | DOI: 10.1210/endocr/bqab197

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by elevated androgens and anti-Mullerian hormone (AMH). These hormones remain elevated throughout pregnancy, and potential effects of hormone exposure on offspring from women with PCOS remain largely unexplored. Expanding on recent reports of prenatal AMH exposure in mice, we have fully characterized the reproductive consequences of prenatal AMH (pAMH) exposure throughout the lifespan of first- and second-generation offspring of both sexes. We also sought to elucidate mechanisms underlying pAMH-induced reproductive effects. There is a known reciprocal relationship between AMH and androgens, and in PCOS and PCOS-like animal models, androgen feedback is dysregulated at the level the hypothalamus. Kisspeptin neurons express androgen receptors and play a critical role in sexual development and function. We therefore hypothesized that pAMH-induced reproductive phenotypes would be mediated by androgen signaling at the level of kisspeptin cells. We tested the pAMH model in kisspeptin-specific androgen receptor knockout (KARKO) mice and found that virtually all pAMH-induced phenotypes assayed are eliminated in KARKO offspring compared to littermate controls. By demonstrating the necessity of androgen receptor in kisspeptin cells to induce pAMH phenotypes, we have advanced understanding of the interactions between AMH and androgens in the context of prenatal exposure, which could have significant implications for children of women with PCOS.

Genetic predisposition to tinnitus in the UK Biobank population

Scientific reports

2021 Sep 13

Urbanek, ME;Zuo, J;
PMID: 34518561 | DOI: 10.1038/s41598-021-97350-z

Tinnitus, the phantom perception of noise originating from the inner ear, has been reported by 15% of the world's population, with many patients reporting major deficits to cognition and mood. However, both objective diagnostic tools and targeted therapeutic strategies have yet to be established. To better understand the underlying genes that may preclude tinnitus, we performed a genome-wide association study of the UK Biobank's 49,960 whole exome sequencing participants to identify any loci strongly associated with tinnitus. We identified 17 suggestive single nucleotide polymorphisms (p < 1e-5) spanning 13 genes in two sex-separated cohorts reporting chronic, bothersome tinnitus (control males n = 7,315, tinnitus males n = 226, control females n = 11,732, tinnitus females n = 300). We also found a significant missense mutation in WDPCP (p = 3.959e-10) in the female cohort, a mutation which has been previously implicated in typical neuronal functioning through axonal migration and structural reinforcement, as well as in Bardet-Biedl syndrome-15, a ciliopathy. Additionally, in situ hybridization in the embryonic and P56 mouse brain demonstrated that the majority of these genes are expressed within the dorsal cochlear nucleus, the region of the brain theorized to initially induce tinnitus. Further RT-qPCR and RNAScope data also reveals this expression pattern. The results of this study indicate that predisposition to tinnitus may span across multiple genomic loci and be established by weakened neuronal circuitry and maladaptive cytoskeletal modifications within the dorsal cochlear nucleus.

Mechanisms of vascular smooth muscle cell investment and phenotypic diversification in vascular diseases

Biochemical Society transactions

2021 Sep 08

Worssam, MD;Jørgensen, HF;
PMID: 34495326 | DOI: 10.1042/BST20210138

In contrast with the heart, the adult mammalian vasculature retains significant remodelling capacity, dysregulation of which is implicated in disease development. In particular, vascular smooth muscle cells (VSMCs) play major roles in the pathological vascular remodelling characteristic of atherosclerosis, restenosis, aneurysm and pulmonary arterial hypertension. Clonal lineage tracing revealed that the VSMC-contribution to disease results from the hyperproliferation of few pre-existing medial cells and suggested that VSMC-derived cells from the same clone can adopt diverse phenotypes. Studies harnessing the powerful combination of lineage tracing and single-cell transcriptomics have delineated the substantial diversity of VSMC-derived cells in vascular lesions, which are proposed to have both beneficial and detrimental effects on disease severity. Computational analyses further suggest that the pathway from contractile VSMCs in healthy arteries to phenotypically distinct lesional cells consists of multiple, potentially regulatable, steps. A better understanding of how individual steps are controlled could reveal effective therapeutic strategies to minimise VSMC functions that drive pathology whilst maintaining or enhancing their beneficial roles. Here we review current knowledge of VSMC plasticity and highlight important questions that should be addressed to understand how specific stages of VSMC investment and phenotypic diversification are controlled. Implications for developing therapeutic strategies in pathological vascular remodelling are discussed and we explore how cutting-edge approaches could be used to elucidate the molecular mechanisms underlying VSMC regulation.

Elucidating the Role of Cerebellar Synaptic Dysfunction in C9orf72-ALS/FTD- a Systematic Review and Meta-Analysis

Cerebellum (London, England)

2021 Sep 07

Kaliszewska, A;Allison, J;Col, TT;Shaw, C;Arias, N;
PMID: 34491551 | DOI: 10.1007/s12311-021-01320-0

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) with synaptic dysfunction identified as an early pathological hallmark. Although TDP-43 pathology and overt neurodegeneration are largely absent from the cerebellum, the pathological hallmarks of RNA foci and dipeptide repeat protein (DPR) inclusions are most abundant. Here, we present a systematic literature search in the databases of PubMed, Scopus, Embase, Web of Science and Science Direct up until March 5, 2021, which yielded 19,515 publications. Following the exclusion criteria, 72 articles were included having referred to C9orf72, synapses and the cerebellum. Meta-analyses were conducted on studies which reported experimental and control groups with means and standard deviations extracted from figures using the online tool PlotDigitizer. This revealed dendritic defects (P = 0.03), reduced C9orf72 in human patients (P = 0.005) and DPR-related neuronal loss (P = 0.0006) but no neuromuscular junction abnormalities (P = 0.29) or cerebellar neuronal loss (P = 0.23). Our results suggest that dendritic arborisation defects, synaptic gene dysregulation and altered synaptic neurotransmission may drive cerebellar synaptic dysfunction in C9-ALS/FTD. In this review, we discuss how the chronological appearance of the different pathological hallmarks alters synaptic integrity which may have profound implications for disease progression. We conclude that a reduction in C9orf72 protein levels combined with the accumulation of RNA foci and DPRs act synergistically to drive C9 synaptopathy in the cerebellum of C9-ALS/FTD patients.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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