scholarly journals Novel Loss-of-Function Mutations in DNAH1 Displayed Different Phenotypic Spectrum in Humans and Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Ranjha Khan ◽  
Qumar Zaman ◽  
Jing Chen ◽  
Manan Khan ◽  
Ao Ma ◽  
...  
Keyword(s):  
Male Infertility ◽  
Mutant Mice ◽  
Pathophysiological Mechanism ◽  
Loss Of Function ◽  
Fibrous Sheath ◽  
Transmission Electron Microscopy Analysis ◽  
Central Pair ◽  
Recurrent Mutations ◽  
The Impact

Male infertility is a prevalent disorder distressing an estimated 70 million people worldwide. Despite continued progress in understanding the causes of male infertility, idiopathic sperm abnormalities such as multiple morphological abnormalities of sperm flagella (MMAF) still account for about 30% of male infertility. Recurrent mutations in DNAH1 have been reported to cause MMAF in various populations, but the underlying mechanism is still poorly explored. This study investigated the MMAF phenotype of two extended consanguineous Pakistani families without manifesting primary ciliary dyskinesia symptoms. The transmission electron microscopy analysis of cross-sections of microtubule doublets revealed a missing central singlet of microtubules and a disorganized fibrous sheath. SPAG6 staining, a marker generally used to check the integration of microtubules of central pair, further confirmed the disruption of central pair in the spermatozoa of patients. Thus, whole-exome sequencing (WES) was performed, and WES analysis identified two novel mutations in the DNAH1 gene that were recessively co-segregating with MMAF phenotype in both families. To mechanistically study the impact of identified mutation, we generated Dnah1 mice models to confirm the in vivo effects of identified mutations. Though Dnah1△iso1/△iso1 mutant mice represented MMAF phenotype, no significant defects were observed in the ultrastructure of mutant mice spermatozoa. Interestingly, we found DNAH1 isoform2 in Dnah1△iso1/△iso1 mutant mice that may be mediating the formation of normal ultrastructure in the absence of full-length protein. Altogether we are first reporting the possible explanation of inconsistency between mouse and human DNAH1 mutant phenotypes, which will pave the way for further understanding of the underlying pathophysiological mechanism of MMAF.

scholarly journals Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Susana Beceiro ◽  
Attila Pap ◽  
Zsolt Czimmerer ◽  
Tamer Sallam ◽  
Jose A. Guillén ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Low Density Lipoprotein ◽  
Myeloid Cells ◽  
Density Lipoprotein ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Cd38 Expression ◽  
The Impact

ABSTRACTThe liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migrationin vitroandin vivo. Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR−/−) LDLR−/−mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

scholarly journals Long Term Pharmacological Perturbation of Autophagy in Mice: Are HCQ Injections a Relevant Choice?

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 47 ◽  
Author(s):  
Jean-Daniel Masson ◽  
Benoit Blanchet ◽  
Baptiste Periou ◽  
François-Jérôme Authier ◽  
Baharia Mograbi ◽  
...  
Keyword(s):  
Mouse Models ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Evolutionarily Conserved ◽  
Atg Genes ◽  
Conditional Inhibition ◽  
The Impact ◽  
Catabolic Process

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whose loss-of-function has been linked to a growing list of pathologies. Knockout mouse models of key autophagy genes have been instrumental in the demonstration of the critical functions of autophagy, but they display early lethality, neurotoxicity and unwanted autophagy-independent phenotypes, limiting their applications for in vivo studies. To avoid problems encountered with autophagy-null transgenic mice, we investigated the possibility of disturbing autophagy pharmacologically in the long term. Hydroxychloroquine (HCQ) ip injections were done in juvenile and adult C57bl/6j mice, at range doses adapted from the human malaria prophylactic treatment. The impact on autophagy was assessed by western-blotting, and juvenile neurodevelopment and adult behaviours were evaluated for four months. Quite surprisingly, our results showed that HCQ treatment in conditions used in this study neither impacted autophagy in the long term in several tissues and organs nor altered neurodevelopment, adult behaviour and motor capabilities. Therefore, we recommend for future long-term in vivo studies of autophagy, to use genetic mouse models allowing conditional inhibition of selected Atg genes in appropriate lineage cells instead of HCQ treatment, until it could be successfully revisited using higher HCQ doses and/or frequencies with acceptable toxicity.

scholarly journals Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status

2020 ◽  
Vol 4 (5) ◽  
pp. 845-854 ◽  
Author(s):  
Raksha Shrestha ◽  
Mamiko Sakata-Yanagimoto ◽  
Koichiro Maie ◽  
Motohiko Oshima ◽  
Masatomo Ishihara ◽  
...  
Keyword(s):  
Median Survival ◽  
Myeloid Leukemia ◽  
Recurrent Events ◽  
Loss Of Function ◽  
Genetic Event ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
The Impact

Abstract Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

scholarly journals Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Anne Rovelet-Lecrux ◽  
Sebastien Feuillette ◽  
Laetitia Miguel ◽  
Catherine Schramm ◽  
Ségolène Pernet ◽  
...  
Keyword(s):  
3D Structure ◽  
Hek293 Cells ◽  
Pathophysiological Mechanism ◽  
Loss Of Function ◽  
Missense Variants ◽  
Major Player ◽  
Functional Consequences ◽  
Endosomal System ◽  
The Impact ◽  
Clear Increase

AbstractThe SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer’s disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

scholarly journals Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3

2020 ◽  
Vol 295 (18) ◽  
pp. 6007-6022 ◽  
Author(s):  
József Jászai ◽  
Kristina Thamm ◽  
Jana Karbanová ◽  
Peggy Janich ◽  
Christine A. Fargeas ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Photoreceptor Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Loss Of Function ◽  
Animal Kingdom ◽  
Left Right Asymmetry ◽  
And Function ◽  
The Impact

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

scholarly journals Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

2019 ◽  
Vol 3 (15) ◽  
pp. 2355-2367 ◽  
Author(s):  
Yueh-Chwen Hsu ◽  
Tsung-Chih Chen ◽  
Chien-Chin Lin ◽  
Chang-Tsu Yuan ◽  
Chia-Lang Hsu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Knockout Mice ◽  
Neurodevelopmental Disorder ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
The Impact

Abstract Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

scholarly journals Jointly reduced inhibition and excitation underlies circuit-wide changes in cortical processing in Rett syndrome

2016 ◽  
Vol 113 (46) ◽  
pp. E7287-E7296 ◽  
Author(s):  
Abhishek Banerjee ◽  
Rajeev V. Rikhye ◽  
Vincent Breton-Provencher ◽  
Xin Tang ◽  
Chenchen Li ◽  
...  
Keyword(s):  
Rett Syndrome ◽  
Time Course ◽  
Pyramidal Neurons ◽  
Reversal Potential ◽  
Mutant Mice ◽  
Loss Of Function ◽  
Cortical Circuits ◽  
Recombinant Human Insulin ◽  
Cortical Pyramidal Neurons

Rett syndrome (RTT) arises from loss-of-function mutations in methyl-CpG binding protein 2 gene (Mecp2), but fundamental aspects of its physiological mechanisms are unresolved. Here, by whole-cell recording of synaptic responses in MeCP2 mutant mice in vivo, we show that visually driven excitatory and inhibitory conductances are both reduced in cortical pyramidal neurons. The excitation-to-inhibition (E/I) ratio is increased in amplitude and prolonged in time course. These changes predict circuit-wide reductions in response reliability and selectivity of pyramidal neurons to visual stimuli, as confirmed by two-photon imaging. Targeted recordings reveal that parvalbumin-expressing (PV+) interneurons in mutant mice have reduced responses. PV-specific MeCP2 deletion alone recapitulates effects of global MeCP2 deletion on cortical circuits, including reduced pyramidal neuron responses and reduced response reliability and selectivity. Furthermore, MeCP2 mutant mice show reduced expression of the cation-chloride cotransporter KCC2 (K+/Cl− exporter) and a reduced KCC2/NKCC1 (Na+/K+/Cl− importer) ratio. Perforated patch recordings demonstrate that the reversal potential for GABA is more depolarized in mutant mice, but is restored by application of the NKCC1 inhibitor bumetanide. Treatment with recombinant human insulin-like growth factor-1 restores responses of PV+ and pyramidal neurons and increases KCC2 expression to normalize the KCC2/NKCC1 ratio. Thus, loss of MeCP2 in the brain alters both excitation and inhibition in brain circuits via multiple mechanisms. Loss of MeCP2 from a specific interneuron subtype contributes crucially to the cell-specific and circuit-wide deficits of RTT. The joint restoration of inhibition and excitation in cortical circuits is pivotal for functionally correcting the disorder.

scholarly journals The METTL3/MALAT1/PTBP1/USP8/TAK1 axis promotes pyroptosis and M1 polarization of macrophages and contributes to liver fibrosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Bo Shu ◽  
Ying-Xia Zhou ◽  
Hao Li ◽  
Rui-Zhi Zhang ◽  
Chao He ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Rna Stability ◽  
Interferon Γ ◽  
In Vivo Model ◽  
Loss Of Function ◽  
M1 Polarization ◽  
The Impact

AbstractPro-inflammatory M1 macrophages, via activating hepatic stellate cells, contribute to liver fibrosis. In this study, we examined the mechanism and the significance of a signaling axis, METTL3/MALAT1/PTBP1/USP8/TAK1, in regulating pyroptosis and M1 polarization of hepatic macrophages. Liver fibrosis model was established in vivo by CCl4 treatment; M1 polarization was induced in vitro by treating macrophages with lipopolysaccharide or interferon γ. Expressions of METTL3, MALAT1, PTBP1, USP8, and TAK1 were measured by RT-PCR and/or Western blot in Kupffer cells (KCs) isolated from in vivo model or in vitro activated macrophages. Macrophage phenotypes including inflammation (RT-qPCR analysis of a panel of proinflammatory cytokines and ELISA on productions of interleukin (IL)−1β and IL-18) and pyroptosis (Western blot of NLRP3, Caspase-1, and GSDMD) were investigated. The impact of METTL3 on m6A methylation of MALAT1 was examined by methylated RNA immunoprecipitation (RIP), the interaction between PTBP1 and MALAT1 or USP8 mRNA by combining RNA pull-down, RIP, and RNA stability assays, and the crosstalk between USP8 and TAK1 by co-immunoprecipitation and protein degradation assays. Functional significance of individual component of METTL3/MALAT1/PTBP1/USP8/TAK1 axis was assessed by combining gain-of-function and loss-of-function approaches. In KCs isolated from in vivo liver fibrosis model or in vitro M1-polarized macrophages, METTL3 was up-regulated, and sequentially, it increased MALAT1 level via m6A methylation, which promoted USP8 mRNA degradation through the interaction with PTBP1. Reduced USP8 expression regulated the ubiquitination and protein stability of TAK1, which promoted pyroptosis and inflammation of macrophages. The signaling cascade METTL3/MALAT1/PTBP1/USP8/TAK1, by essentially stimulating pyroptosis and inflammation of macrophages, aggravates liver fibrosis. Therefore, targeting individual components of this axis may benefit the treatment of liver fibrosis.

scholarly journals Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

2021 ◽  
Author(s):  
Anne Rovelet-Lecrux ◽  
Sebastien Feuillette ◽  
Laetitia Miguel ◽  
Catherine Schramm ◽  
Segolene Pernet ◽  
...  
Keyword(s):  
Protein Structure ◽  
Alzheimer Disease ◽  
Hek293 Cells ◽  
Pathophysiological Mechanism ◽  
Loss Of Function ◽  
Missense Variants ◽  
Major Player ◽  
Functional Consequences ◽  
Sorl1 Gene ◽  
The Impact

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of SorLA protein. An initial screening, based on the overexpression of 71 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variations (R332W, S577P, and R654W) and two maturation-competent variations (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs were associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Finally, structural analysis of the impact of missense variations on SorLA protein structure indicated that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein structure resulting from changes in the interatomic interactions.

scholarly journals Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice

Gut ◽  
2018 ◽  
Vol 68 (2) ◽  
pp. 301-312 ◽  
Author(s):  
Eszter Hegyi ◽  
Miklós Sahin-Tóth
Keyword(s):  
Chronic Pancreatitis ◽  
Er Stress ◽  
Digestive Enzyme ◽  
Pancreatic Disease ◽  
Mutant Mice ◽  
Inflammatory Disorder ◽  
Loss Of Function ◽  
Stress Markers ◽  
Elevated Expression

ObjectiveChronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme carboxypeptidase A1, were described in sporadic early onset cases and in hereditary pancreatitis. Mutation-induced misfolding of CPA1 and associated endoplasmic reticulum (ER) stress was suggested as potential disease mechanism; however, in vivo evidence has been lacking. The objective of the present study was to create a mouse model that recapitulates features of CPA1-associated chronic pancreatitis.DesignWe knocked-in the most frequently occurring p.N256K human CPA1 mutation to the mouse Cpa1 locus. Mutant mice were characterised with respect to pancreas pathology and ER stress and compared with C57BL/6N and CPA1 null control mice.ResultsIn the CPA1 N256K mutant mice, we observed hallmarks of chronic pancreatitis that included progressive acinar cell atrophy, inflammatory cell infiltration, fibrosis and acinar-ductal metaplasia. In contrast, similarly to the C57BL/6N mice, the CPA1 null control strain exhibited no signs of pancreatic disease. Mutation p.N256K induced misfolding of mouse CPA1 and resulted in elevated expression of ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) both in cell culture and mutant mice.ConclusionThe results offer categorical evidence that CPA1 mutations elicit enzyme misfolding and cause chronic pancreatitis via an ER stress-related mechanism.

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