Atp8a1 deletion increases the proliferative activity of hematopoietic stem cells by impairing PTEN function.

PURPOSE: The eukaryotic cell plasma membrane contains several asymmetrically distributed phospholipids, which is maintained by the P4-ATPase flippase complex. Herein, we demonstrated the biological effects and mechanisms of asymmetrical loss in hematopoietic stem cells (HSCs). METHODS: An Atp8a1 knockout mouse model was employed, from which the HSC (long-term HSCs and short-term HSCs) population was analyzed to assess their abundance and function. Additionally, competitive bone marrow transplantation and 5-FU stress assays were performed. RNA sequencing was performed on Hematopoietic Stem and Progenitor Cells, and DNA damage was assayed using immunofluorescence staining and comet electrophoresis. The protein abundance for members of key signaling pathways was confirmed using western blotting. RESULTS: Atp8a1 deletion resulted in slight hyperleukocytosis, associated with the high proliferation of HSCs and BCR/ABL1 transformed leukemia stem cells (LSCs). Atp8a1 deletion increased the repopulation capability of HSCs with a competitive advantage in reconstitution assay. HSCs without Atp8a1 were more sensitive to 5-FU-induced apoptosis. Moreover, Atp8a1 deletion prevented HSC DNA damage and facilitated DNA repair processes. Genes involved in PI3K-AKT-mTORC1, DNA repair, and AP-1 complex signaling were enriched and elevated in HSCs with Atp8a1 deletion. Furthermore, Atp8a1 deletion caused decreased PTEN protein levels, resulting in the activation of PI3K-AKT-mTORC1 signaling, further increasing the activity of JNK/AP-1 signaling and YAP1 phosphorylation. CONCLUSION: We identified the role of Atp8a1 on hematopoiesis and HSCs. Atp8a1 deletion resulted in the loss of phosphatidylserine asymmetry and intracellular signal transduction chaos.

A missense mutation in Pitx2 leads to early-onset glaucoma via NRF2-YAP1 axis.

Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.

Loss of phosphatidylserine flippase ß-subunit Tmem30a in podocytes leads to albuminuria and glomerulosclerosis.

The asymmetric distribution of phosphatidylserine (PS) in the cytoplasmic leaflet of eukaryotic cell plasma membranes is regulated by a group of P4-ATPases (named PS flippases) and the ß-subunit TMEM30A. Podocytes in the glomerulus form a filtration barrier to prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy. We observed reduced TMEM30A expression in patients with minimal change disease and membranous nephropathy, indicating potential roles of TMEM30A in podocytopathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (KO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis. Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpointing the mechanism of podocyte damage and potential therapeutic targets.

The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis.

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of ß-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced ß-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/ß-catenin signaling.

The phosphatidylserine flippase ß-subunit Tmem30a is essential for normal insulin maturation and secretion.

The processing, maturation, and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the ß subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet ß cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the plasma membrane (PM), which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis and suggest the importance of membrane phospholipid distribution in metabolic disorders.

Deletion of Asrgl1 Leads to Photoreceptor Degeneration in Mice.

The asparaginase and isoaspartyl peptidase 1 (ASRGL1) is an L-asparaginase and beta-aspartyl peptidase enzyme that may be involved in the formation of L-aspartate, a neurotransmitter that can operate as an excitatory neurotransmitter in some brain regions. Although variants in ASRGL1 have been reported in retinitis pigmentosa (RP) patients, the in vivo functions and mechanisms of ASRGL in RP remains unknown due to the lack of suitable disease models. To explore the role of ASRGL in RP, we generated an Asrgl1 knockout mouse model (Asrgl1 KO) using the CRISPR/Cas9 technique. Asrgl1 ablation in mice led to an attenuated electroretinogram (ERG) response around 8 months. The thickness of the outer nuclei layer (ONL) started to decrease around 9 months in Asrgl1 KO mice and gradually intensified at 12 and 15 months. Immunostaining revealed thinner inner segment (IS) and thinner outer segment (OS) as well as the progressive degeneration of rod and cone cells in Asrgl1 KO mice. One hundred forty-nine transcriptional differentially expressed genes (DEGs) were found by RNA-seq in Asrgl1 KO retina. These DEGs were linked to a number of biological processes that were considerably enriched, including gastrointestinal disease and organismal injury and abnormalities. By analysis of canonical pathways, glucocorticoid receptor signaling was the most significant canonical pathway altered in Asrgl1 KO retina. Several molecules, including NFE2L2, IL-4, Foxp3, and Fos, were in the central nodes of the interaction network in Asrgl1 KO retina. In summary, our study provided a knockout mouse model for a better understanding of the molecular mechanism for ASRGL1-related RP.

Identification of Novel EYS Mutations by Targeted Sequencing Analysis.

Purpose: Retinitis pigmentosa (RP) is an inherited and progressive degenerative retinal disease that often results in severe vision loss and blindness. However, mutations in known RP disease genes account for only 60% of RP cases, indicating that there are additional pathogenic mutations are yet to be identified. We aimed to identify the causative mutations in the eyes shut homolog (EYS) gene in a cohort of Chinese RP and rod-cone dystrophy families. Materials and Methods: Targeted next-generation sequencing was applied to identify novel mutations in these patients. Candidate variants were evaluated using bioinformatics tools. Mutations were confirmed by Sanger sequencing. Results: We identified eight heterozygous mutations in the EYS gene in the four probands, including a novel frameshift deletion mutation, c.8242_8243del (p.L2748fs); a novel insertion mutation, c.5802_5803insT (p.I1935YfsX6); a novel splicing mutation, c.1300-1G>A; two heterozygous stop-gain mutations, c.1750G>T (p.E584X) and c.8805C>A (p.Y2935X); and three novel missense mutations, c.8269G>A (p.V2757I), c.2545C>T (p.R849C) and c.7506C>A (p.S2502R). Only c.8805C>A had been reported previously in RP patients. None of these mutations were present in 1000 control individuals. Conclusions: We identified seven novel mutations in the EYS gene, expanding the mutational specra of EYS in Chinese patients with RP and rod-cone dystrophy.

Loss of the ER membrane protein complex subunit Emc3 leads to retinal bipolar cell degeneration in aged mice.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved protein complex involved in inserting the transmembrane domain of membrane proteins into membranes in the ER. EMC3 is an essential component of EMC and is important for rhodopsin synthesis in photoreceptor cells. However, the in vivo function of Emc3 in bipolar cells (BCs) has not been determined. To explore the role of Emc3 in BCs, we generated a BC-specific Emc3 knockout mouse model (named Emc3 cKO) using the Purkinje cell protein 2 (Pcp2) Cre line. Although normal electroretinography (ERG) b-waves were observed in Emc3 cKO mice at 6 months of age, Emc3 cKO mice exhibited reduced b-wave amplitudes at 12 months of age, as determined by scotopic and photopic ERG, and progressive death of BCs, whereas the ERG a-wave amplitudes were preserved. PKCa staining of retinal cryosections from Emc3 cKO mice revealed death of rod BCs. Loss of Emc3 led to the presence of the synaptic protein mGLuR6 in the outer nuclear layer (ONL). Immunostaining analysis of presynaptic protein postsynaptic density protein 95 (PSD95) revealed rod terminals retracted to the ONL in Emc3 cKO mice at 12 months of age. In addition, deletion of Emc3 resulted in elevated glial fibrillary acidic protein, indicating reactive gliosis in the retina. Our data demonstrate that loss of Emc3 in BCs leads to decreased ERG response, increased astrogliosis and disruption of the retinal inner nuclear layer in mice of 12 months of age. Taken together, our studies indicate that Emc3 is not required for the development of BCs but is important for long-term survival of BCs.

Disease Mutation Study Identifies Critical Residues for Phosphatidylserine Flippase ATP11A.

Phosphatidylserine flippase (P4-ATPase) transports PS from the outer to the inner leaflet of the lipid bilayer in the membrane to maintain PS asymmetry, which is important for biological activities of the cell. ATP11A is expressed in multiple tissues and plays a role in myotube formation. However, the detailed cellular function of ATP11A remains elusive. Mutation analysis revealed that I91, L308, and E897 residues in ATP8A2 are important for flippase activity. In order to investigate the roles of these corresponding amino acid residues in ATP11A protein, we assessed the expression and cellular localization of the respective ATP11A mutant proteins. ATP11A mainly localizes to the Golgi and plasma membrane when coexpressed with the ß-subunit of the complex TMEM30A. Y300F mutation causes reduced ATP11A expression, and Y300F and D913K mutations affect correct localization of the Golgi and plasma membrane. In addition, Y300F and D913K mutations also affect PS flippase activity. Our data provides insight into important residues of ATP11A.

Identification of novel USH2A mutations in patients with autosomal recessive retinitis pigmentosa via targeted next­generation sequencing.

Retinitis pigmentosa (RP) is a group of inheritable blindness retinal diseases characterized by the death of photoreceptor cells and a gradual loss of peripheral vision. Mutations in Usher syndrome type 2 (USH2A) have been reported in RP with or without hearing loss. The present study aimed to identify causative mutations in a cohort of families with RP from China. A cohort of 62 non­syndromic families with RP and 30 sporadic cases were enrolled in this study. All affected members underwent a complete ophthalmic examination, including fundus photography, visual­field test and optical coherence tomography examination. Next­generation sequencing­targeted sequencing of 163 genes involved in inheritable retinal disorders was performed on the probands. Stringent bioinformatics data analysis was applied to identify potential candidate variants. In total, 6 novel mutations and 2 known mutations of USH2A were identified in 4 families with RP. A stop­gain mutation (c.C1731A) and a missense mutation (c.G8254A) were identified in RP family RP­2148. In another RP family, RP­2150, a known mutation (c.G802A) and a novel frameshift insertion mutation (c.12086dupA) were discovered. A novel stop­gain mutation (c.G11754A) and a missense mutation (c.G13465A) were identified in family rpz05. A novel missense mutation (c.C9328G) and a known missense mutation (c.G8232C) were also identified. These mutations were subsequently confirmed by Sanger sequencing. All 6 novel mutations affected highly conserved amino acid residues, and were absent in 1,000 ethnically matched controls. Taken together, the present study has reported on 6 novel USH2A mutations in 4 families with RP, and has expanded the mutation spectrum of USH2A in autosomal recessive RP in the Chinese population, thus providing important information for the molecular diagnosis and screening of RP.

Deletion of the Impg2 gene causes the degeneration of rod and cone cells in mice.

Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.

A moderated mediation model of perceived stress, negative emotions and mindfulness on fertility quality of life in women with recurrent pregnancy loss.

PURPOSE: This study aimed to investigate the level of fertility related quality of life (FertiQoL) in women with recurrent pregnancy loss (RPL), and to further examine moderated mediation effects of dispositional mindfulness and negative emotions on the relationship between perceived stress and FertiQoL. METHODS: A cross-sectional study was conducted with 262 participants recruited from the infertility outpatient clinics. A self-administered, structured questionnaire including the Simplified Chinese version of FertiQoL tool, the Perceived Stress Scale-10 (PSS-10), the Chinese Version of the Hospital Anxiety and Depression Scale (HADS), and the Mindfulness Attention Awareness Scale (MAAS) was used to collect information in this research. The mediation model and moderated mediation model were conducted using the PROCESS macro for SPSS. RESULTS: The mean score of Core FertiQoL was 64.59 (SD = 14.76) among women with RPL. Mediation analysis revealed that the association between perceived stress and FertiQoL was partially mediated by negative emotions (indirect effect = - 0.194 for anxiety, and - 0.151 for depression, all P < 0.001). Moderated mediation analysis indicated that the indirect effects of perceived stress on FertiQoL through negative emotions were significantly moderated by dispositional mindfulness. Specifically, the indirect effects of perceived stress on FertiQoL through negative emotions decreased were significant as dispositional mindfulness levels increased. CONCLUSION: Overall, women with RPL experienced poor FertiQoL. The Moderated mediation model provides a better understanding of how perceived stress, negative emotions and dispositional mindfulness work together to affect FertiQoL. Interventions aiming to improve FertiQoL in women with RPL should consider targeting these aspects.

Configuration-Controllable E/Z Isomers Based on Tetraphenylethene: Synthesis, Characterization, and Applications.

Configuration-controllable E/Z isomers based on tetraphenylethene were prepared with a facile and effective method. First, compounds 1 and 2, configuration-controllable precursors of E/Z isomers, were synthesized. Then, pure E/Z isomers were obtained via Suzuki reaction, avoiding the difficulties of separation. The conformational changes of E/Z isomers can occur through photoactivation. Importantly, red-shifts of 66 nm from 6 (E-) to 3 (Z-) and 58 nm from 7 (E-) to 4 (Z-) were observed remarkably on the photoluminescence (PL) emission spectra. The Z isomer showed a longer fluorescence lifetime compared with the E isomer. The Z isomers 3 and 4 exhibited piezofluorochromism under grinding, whereas the E isomers 6 and 7 showed no such behaviors. The E isomer has better thermal stability than the Z isomer. Lastly, graphene-like molecules were synthesized with the FeCl3/CH3NO2 system. The E and Z isomers after oxidation showed negligible differences in the PL emission spectra because the effective conjugated lengths of oxidized E and Z isomers were both extended. Furthermore, the fabricated field-effect transistors showed nice performance with mobilities of 0.92 and 1.14 cm-2 V-1 s-1 at low operating voltages, respectively.