Silencing of Activity During Hypoxia Improves Functional Outcomes in Motor Neuron Networks in vitro

The effects of hypoxia, or reduced oxygen supply, to brain tissue can be disastrous, leading to extensive loss of function. Deoxygenated tissue becomes unable to maintain healthy metabolism, which leads to increased production of reactive oxygen species (ROS) and loss of calcium homoeostasis, with damaging downstream effects. Neurons are a highly energy demanding cell type, and as such they are highly sensitive to reductions in oxygenation and some types of neurons such as motor neurons are even more susceptible to hypoxic damage. In addition to the immediate deleterious effects hypoxia can have on neurons, there can be delayed effects which lead to increased risk of developing neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), even if no immediate consequences are apparent. Furthermore, impairment of the function of various hypoxia-responsive factors has been shown to increase the risk of developing several neurodegenerative disorders. Longitudinal assessment of electrophysiological network activity is underutilised in assessing the effects of hypoxia on neurons and how their activity and communication change over time following a hypoxic challenge. This study utilised multielectrode arrays and motor neuron networks to study the response to hypoxia and the subsequent development of the neuronal activity over time, as well as the effect of silencing network activity during the hypoxic challenge. We found that motor neuron networks exposed to hypoxic challenge exhibited a delayed fluctuation in multiple network activity parameters compared to normoxic networks. Silencing of activity during the hypoxic challenge leads to maintained bursting activity, suggesting that functional outcomes are better maintained in these networks and that there are activity-dependent mechanisms involved in the network damage following hypoxia.

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Biopsychosocial Patterning of Multimorbidity and Its Consequences

The Oxford Handbook of Integrative Health Science ◽

10.1093/oxfordhb/9780190676384.013.14 ◽

2018 ◽

pp. 220-236

Author(s):

Elliot Friedman ◽

Beth LeBreton ◽

Lindsay Fuzzell ◽

Elizabeth Wehrpsann

Keyword(s):

Quality Of Life ◽

Older Adults ◽

Functional Outcomes ◽

The United States ◽

Chronic Medical Conditions ◽

Quality Of Life Research ◽

Increased Risk ◽

Over Time

By many estimates the majority of adults over age 65 have two or more chronic medical conditions (multimorbidity) and are consequently at increased risk of adverse functional outcomes. Nonetheless, many older adults with multimorbidity are able to maintain high levels of function and retain good quality of life. Research presented here is designed to understand the influences that help ensure better functional outcomes in these older adults. This chapter presents findings that draw on data from the Midlife in the United States study. The independent and interactive contributions of diverse factors to multimorbidity and changes in multimorbidity over time are reviewed. The degree that multimorbidity increases risk of cognitive impairment and disability is examined. The role of inflammation as a mediator is considered. Multimorbidity is increasingly the norm for older adults, so better understanding of factors contributing to variability in multimorbidity-related outcomes can lead to improved quality of life.

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CIC missense variants contribute to susceptibility for spina bifida

10.22541/au.163826470.06627349/v1 ◽

2021 ◽

Author(s):

Xiao Han ◽

Xuanye Cao ◽

Vanessa Aguiar-Pulido ◽

Wei Yang ◽

Menuka Karki ◽

...

Keyword(s):

Spina Bifida ◽

Cell Line ◽

Neural Tube ◽

Folate Receptor ◽

Spinocerebellar Ataxia Type ◽

Loss Of Function ◽

Missense Variants ◽

Increased Risk ◽

Cell Line Hela

Neural Tube Defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to cerebral folate deficiency by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole genome sequencing (WGS) data of 140 isolated spina bifida cases and identified 8 missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants down regulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

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GFI1 Is a Tumor Suppressor in Myeloid Progenitors

Blood ◽

10.1182/blood.v112.11.2942.2942 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2942-2942

Author(s):

Aditya Chaubey ◽

Shane Hormon ◽

Chinavenmeni S. Velu ◽

Tristan Bourdeau ◽

Jinfang Zhu ◽

...

Keyword(s):

Dna Sequences ◽

Myeloid Leukemia ◽

Dependent Manner ◽

Loss Of Function ◽

Increased Risk ◽

Myeloid Leukemias ◽

Dose Dependent ◽

Anterior Posterior

Abstract In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.

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Genetic Analysis of Osteoblast Activity Identifies Zbtb40 as a Regulator of Osteoblast Activity and Bone Mass

10.1101/828525 ◽

2019 ◽

Author(s):

Madison L. Doolittle ◽

Gina M Calabrese ◽

Larry D. Mesner ◽

Dana A. Godfrey ◽

Robert D. Maynard ◽

...

Keyword(s):

Bone Formation ◽

Genetic Basis ◽

Association Studies ◽

Genome Wide Association Studies ◽

Loss Of Function ◽

Mineral Density ◽

Osteoblast Activity ◽

Genome Wide ◽

Increased Risk

ABSTRACTOsteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

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16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro

Nature Communications ◽

10.1038/s41467-021-23113-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maria Sundberg ◽

Hannah Pinson ◽

Richard S. Smith ◽

Kellen D. Winden ◽

Pooja Venugopal ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Wide Spectrum ◽

Copy Number Variations ◽

Network Activity ◽

Neuron Networks ◽

Soma Size ◽

Marker Expression ◽

Induced Pluripotent ◽

Human Ipsc

AbstractReciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.

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A rare variant on a common risk haplotype of HFE causes increased risk of hereditary hemochromatosis

10.1101/547471 ◽

2019 ◽

Author(s):

Andrew M. Glazer ◽

Lisa Bastarache ◽

Lynn Hall ◽

Laura Short ◽

Tiffany Shields ◽

...

Keyword(s):

Cell Surface ◽

Hereditary Hemochromatosis ◽

Iron Complex ◽

Risk Scores ◽

Risk Haplotype ◽

Loss Of Function ◽

Double Mutation ◽

Variant Of Uncertain Significance ◽

Increased Risk

AbstractHereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The most common form, HH1, is caused by loss of function variants in HFE. HFE encodes a cell surface protein that binds to the Transferrin Receptor (TfR1), reducing TfR1’s affinity for the transferrin/iron complex and thereby limiting cellular iron uptake. Two common missense alleles for HH1 have been identified, HFE C282Y and HFE H63D; H63D is considered to be a less penetrant allele. When we deployed Phenotype Risk Scores (PheRS), a method that aggregates multiple symptoms together in Electronic Health Records (EHRs), we identified HFE E168Q as a novel variant associated with HH. E168Q is on the same haplotype as H63D, and in a crystal structure HFE E168 lies at the interface of the HFE-TfR1 interaction and makes multiple salt bridge connections with TfR1. In in vitro cell surface abundance experiments, the HFE E168Q+H63D double mutation surprisingly increased cell surface abundance of HFE by 10-fold compared to wildtype. In coimmunoprecipitation experiments, however, HFE C282Y, E168Q, and E168Q+H63D completely abolished the interaction between HFE and TfR1, while H63D alone only partially reduced binding. These findings provide mechanistic insight to validate the PheRS result that HFE E168Q is an HH1-associated allele and lead to the reclassification of E168Q from a variant of uncertain significance to a pathogenic variant, according to ACMG guidelines. HFE E168Q results in loss of HFE function by disrupting the HFE-TfR1 interaction. In addition, some disease manifestations attributed to H63D may reflect the functional effects of E168Q.

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Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905690116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18429-18434 ◽

Cited By ~ 13

Author(s):

Kyoko Chiba ◽

Hironori Takahashi ◽

Min Chen ◽

Hiroyuki Obinata ◽

Shogo Arai ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neuron Disease ◽

Synaptic Vesicle ◽

Axonal Transport ◽

Point Mutations ◽

Loss Of Function ◽

In Vitro Motility ◽

Anterograde Axonal Transport ◽

Abnormal Accumulation

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations inKIF1Aare associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for howKIF1Amutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found thatKIF1Amutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into theCaenorhabditis elegans KIF1Ahomologunc-104revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

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Tet2 Deficiency Rejuvenates Hematopoietic Stem and Progenitor Cells during Ageing

Blood ◽

10.1182/blood-2020-141705 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-37

Author(s):

Tingting Hong ◽

Shengli Li ◽

Shaohai Fang ◽

Anna Guzman ◽

Wei Han ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Pool Size ◽

Loss Of Function ◽

Self Renewal ◽

Hematopoietic Stem ◽

Increased Risk ◽

Age Dependent

Ageing is accompanied by a significant reduction of hematopoietic competence driven by various causes including epigenetic alternations [1-4]. Ten-eleven translocation 2 (TET2) has been well delineated as a critical epigenetic regulator that affects hematopoietic progenitor and stem cells (HSPCs) function. Tet2 deficiency confers advantages in clonal expansion of HSPCs and skews myeloid lineage differentiation, giving rise to increased risk of hematological malignancy transformation [5-8]. TET2 loss-of-function mutations are frequently detected in aged HSPCs [10-11], thereby raising the question of how Tet2 deficiency affects HSPCs self-renewal and lineage specification during ageing. To address this question, we harvested HSPCs from wild-type (WT) or Tet2KO young and aged donor mice, followed by competitive bone marrow transplantations to monitor age-dependent functional alterations. Despite the enlargement of the HSC pool size (the number of cells with regenerative potential) in aged mice, the aged WT HSPCs exhibited lower self-renewal capability and displayed impaired hematopoietic differentiation when competed against young stem cells. However, we found that both aged and young Tet2-deficient HSPCs shared comparable peripheral blood reconstitution, indicating no engraftment defects were caused by age for Tet2-deficient HSPCs. In parallel, scRNA-seq analysis revealed that Tet2 deficiency and age promoted the expansion of HSC compartment in a synergistic manner, leading to the largely augmented pool size of Tet2-deficient aged HSCs. But unlike aged WT stem cells, these expanded aged Tet2-null stem cells retained high self-renewal potential and possessed a competitive advantage of lineage outputs both in vitro and in vivo. Overall, through conducting repopulation assays and single-cell transcriptomes analysis, we have demonstrated that Tet2 ablation alters age-dependent HSC functional decline, revealing a disparate ageing process in the Tet2-deficient haemopoietic system. Disclosures No relevant conflicts of interest to declare.

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Testosterone-induced increase in libido in a patient with a loss-of-function mutation in the AR gene

Endocrinology Diabetes and Metabolism Case Reports ◽

10.1530/edm-21-0031 ◽

2021 ◽

Vol 2021 ◽

Author(s):

Laura Marino ◽

Andrea Messina ◽

James S Acierno ◽

Franziska Phan-Hug ◽

Nicolas J Niederländer ◽

...

Keyword(s):

Androgen Receptor ◽

Androgen Insensitivity Syndrome ◽

Surgical Removal ◽

List Type ◽

Loss Of Function ◽

Testosterone Treatment ◽

Androgen Insensitivity ◽

Increased Risk ◽

Loss Of Libido

Summary Complete androgen-insensitivity syndrome (CAIS), a disorder of sex development (46,XY DSD), is caused primarily by mutations in the androgen receptor (AR). Gonadectomy is recommended due to the increased risk of gonadoblastoma, however, surgical intervention is often followed by loss of libido. We present a 26-year-old patient with CAIS who underwent gonadectomy followed by a significant decrease in libido, which was improved with testosterone treatment but not with estradiol. Genetic testing was performed and followed by molecular characterization. We found that this patient carried a previously unidentified start loss mutation in the androgen receptor. This variant resulted in an N-terminal truncated protein with an intact DNA binding domain and was confirmed to be loss-of-function in vitro. This unique CAIS case and detailed functional studies raise intriguing questions regarding the relative roles of testosterone and estrogen in libido, and in particular, the potential non-genomic actions of androgens. Learning points N-terminal truncation of androgen receptor can cause androgen-insensitivity syndrome. Surgical removal of testosterone-producing gonads can result in loss of libido. Libido may be improved with testosterone treatment but not with estradiol in some forms of CAIS. A previously unreported AR mutation – p.Glu2_Met190del (c.2T>C) – is found in a CAIS patient and results in blunted AR transcriptional activity under testosterone treatment.

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Paradigmatic De Novo GRIN1 Variants Recapitulate Pathophysiological Mechanisms Underlying GRIN1-Related Disorder Clinical Spectrum

International Journal of Molecular Sciences ◽

10.3390/ijms222312656 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12656

Author(s):

Ana Santos-Gómez ◽

Federico Miguez-Cabello ◽

Natalia Juliá-Palacios ◽

Deyanira García-Navas ◽

Víctor Soto-Insuga ◽

...

Keyword(s):

Functional Outcomes ◽

De Novo ◽

Clinical Symptoms ◽

Ionotropic Glutamate Receptors ◽

Loss Of Function ◽

Biophysical Properties ◽

Related Disorder ◽

Functional Studies ◽

Spanish Cohort

Background: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present. Methods: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants. Results: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms. Conclusions: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.

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