Defective Maturation Research Articles

The effect of Myo-Inositol supplement on molecular regulation of folliculogenesis, steroidogenesis, and assisted reproductive technique outcomes in patients with polycystic ovarian syndrome.

The mechanism of Myo-Inositol, as an adjuvant, on key signaling pathways related to oocyte maturation, fertilization rate, and embryo quality as well as ovarian steroidogenesis in cumulus cells of PCOS patients, is still unclear. Infertile patients who were candidates for ART cycles were divided into three groups (n = 30 in each group), including group 1: PCOS patients only receiving folic acid, group 2: PCOS patients receiving daily Myo-Inositol combined with folic acid, and a control group (group 3): normal ovulatory women without PCOS receiving only folic acid from 1month prior to IVF cycle until the day of ovum pick up. During the ART procedure, oocytes maturation, fertilization rate, and embryo quality were assessed. The gene expressions of FSHR, LHR, CYP11A1, CYP19A1, 3β-HSD2, and StAR were also analyzed using qRT-PCR. Western blot analysis was performed for the evaluation of AKT, ERK, CREB, and AMPK phosphorylation. Despite equal number of retrieved oocytes, the percentages of MII oocytes, fertilization rate, and embryo quality were found to be significantly higher in group 2 due to the administration of inofolic. The expressions of all the studied genes were significantly higher in the cumulus cells of group 1 compared to the group 2. Higher phosphorylation of ERK1/2 was found in the groups 2 and 3 compared to the group 1. On the other hand, p-Akt has significantly decreased in the group 2 compared to the group 1. Our study provides new insight into the molecular mechanism underlying the positive effect of Myo-Inositol on intrinsic ovarian defects in PCOS, steroidogenesis, oocyte maturation, fertilization rate, and embryo quality.

The Anti-Inflammatory Agent Bindarit Attenuates the Impairment of Neural Development through Suppression of Microglial Activation in a Neonatal Hydrocephalus Mouse Model.

Neonatal hydrocephalus presents with various degrees of neuroinflammation and long-term neurologic deficits in surgically treated patients, provoking a need for additional medical treatment. We previously reported elevated neuroinflammation and severe periventricular white matter damage in the progressive hydrocephalus (prh) mutant which contains a point mutation in the Ccdc39 gene, causing loss of cilia-mediated unidirectional CSF flow. In this study, we identified cortical neuropil maturation defects such as impaired excitatory synapse maturation and loss of homeostatic microglia, and swimming locomotor defects in early postnatal prh mutant mice. Strikingly, systemic application of the anti-inflammatory small molecule bindarit significantly supports healthy postnatal cerebral cortical development in the prh mutant. While bindarit only mildly reduced the ventricular volume, it significantly improved the edematous appearance and myelination of the corpus callosum. Moreover, the treatment attenuated thinning in cortical Layers II-IV, excitatory synapse formation, and interneuron morphogenesis, by supporting the ramified-shaped homeostatic microglia from excessive cell death. Also, the therapeutic effect led to the alleviation of a spastic locomotor phenotype of the mutant. We found that microglia, but not peripheral monocytes, contribute to amoeboid-shaped activated myeloid cells in prh mutants' corpus callosum and the proinflammatory cytokines expression. Bindarit blocks nuclear factor (NF)-kB activation and its downstream proinflammatory cytokines, including monocyte chemoattractant protein-1, in the prh mutant. Collectively, we revealed that amelioration of neuroinflammation is crucial for white matter and neuronal maturation in neonatal hydrocephalus. Future studies of bindarit treatment combined with CSF diversion surgery may provide long-term benefits supporting neuronal development in neonatal hydrocephalus.SIGNIFICANCE STATEMENT In neonatal hydrocephalus, little is known about the signaling cascades of neuroinflammation or the impact of such inflammatory insults on neural cell development within the perinatal cerebral cortex. Here, we report that proinflammatory activation of myeloid cells, the majority of which are derived from microglia, impairs periventricular myelination and cortical neuronal maturation using the mouse prh genetic model of neonatal hydrocephalus. Administration of bindarit, an anti-inflammatory small molecule that blocks nuclear factor (NF)-kB activation, restored the cortical thinning and synaptic maturation defects in the prh mutant brain through suppression of microglial activation. These data indicate the potential therapeutic use of anti-inflammatory reagents targeting neuroinflammation in the treatment of neonatal hydrocephalus.

A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis

AbstractBarth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.

Disrupted Association of Sensory Neurons With Enveloping Satellite Glial Cells in Fragile X Mouse Model.

Among most prevalent deficits in individuals with Fragile X syndrome (FXS) is hypersensitivity to sensory stimuli and somatosensory alterations. Whether dysfunction in peripheral sensory system contributes to these deficits remains poorly understood. Satellite glial cells (SGCs), which envelop sensory neuron soma, play critical roles in regulating neuronal function and excitability. The potential contributions of SGCs to sensory deficits in FXS remain unexplored. Here we found major structural defects in sensory neuron-SGC association in the dorsal root ganglia (DRG), manifested by aberrant covering of the neuron and gaps between SGCs and the neuron along their contact surface. Single-cell RNAseq analyses demonstrated transcriptional changes in both neurons and SGCs, indicative of defects in neuronal maturation and altered SGC vesicular secretion. We validated these changes using fluorescence microscopy, qPCR, and high-resolution transmission electron microscopy (TEM) in combination with computational analyses using deep learning networks. These results revealed a disrupted neuron-glia association at the structural and functional levels. Given the well-established role for SGCs in regulating sensory neuron function, altered neuron-glia association may contribute to sensory deficits in FXS.

Aging entails distinct requirements for Rb at maintaining adult neurogenesis.

Cell cycle proteins play essential roles in regulating embryonic and adult neurogenesis in the mammalian brain. A key example is the Retinoblastoma protein (Rb) whose loss disrupts the whole neurogenic program during brain development, but only results in increased progenitor proliferation in the adult subventricular zone (SVZ) and compromised long-term neuronal survival in the adult olfactory bulb (OB). Whether this holds true of neurogenesis in the aged brain remains unknown. In this study, we find no evidence of irregular proliferation or early commitment defects in the mid-aged (12-month-old) and old-aged (20-month-old) SVZ following tamoxifen-inducible Rb knockout (Rb iKO) in mice. However, we highlight a striking defect in early maturation of Rb-deficient migrating neuroblasts along the rostral migratory stream (RMS), followed by massive decline in neuronal generation inside the aged OB. In the absence of Rb, we also show evidence of incomplete cell cycle re-entry (CCE) along with DNA damage in the young OB, while we find a similar trend towards CCE but no clear signs of DNA damage or neurodegenerative signatures (pTau or Synuclein accumulation) in the aged OB. However, such phenotype could be masked by the severe maturation defect reported above in addition to the natural decline in adult neurogenesis with age. Overall, we show that Rb is required to prevent CCE and DNA damage in adult-born OB neurons, hence maintain neuronal survival. Moreover, while loss of Rb alone is insufficient to trigger seeding of neurotoxic species, this study reveals age-dependent non-monotonic dynamics in regulating neurogenesis by Rb.

3097 – RNA EDITOR-EXONUCLEASE AXIS SCULPTS THE TRANSCRIPTOME DURING TERMINAL ERYTHROID DIFFERENTIATION

Erythropoiesis is a committed process by which erythroid progenitors become mature red blood cells (RBCs). Reticulocytes are terminal-staged, immature RBCs which contain selected residual RNA after enucleation. Normally, reticulocytes are efficiently processed and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated from iPS or CD34+ hematopoietic stem/progenitor cells in vitro, erythroid cells tend to arrest at the reticulocyte stage. This is the major bottleneck for the complete recapitulation of erythropoiesis in vitro, and a hindrance to modeling hemoglobinopathies in cell cultures. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases 7/4, official names are ZCCHC6/11) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is then recognized by exoribonucleases and targeted for decay. We posited that this is the machinery behind RNA degradation that accompanies terminal erythropoiesis. Utilizing both constitutional and erythroid-specific conditional murine knockout models, we found that blood from TUTase Zcchc6 RNA editor knockout embryos indeed exhibit reticulocytosis with a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease, Dis3l2, phenocopied the RNA decay defect of the Zcchc6 KO. Furthermore, knockout models did not show signs of hemolysis suggesting a cell intrinsic and niche-independent role for the TUTase7-Dis3l2 axis in promoting red blood cell maturation. Modulating the expression of this axis holds promise for enhancing in vitro erythropoiesis and modeling of hemoglobinopathies. Future mechanism studies will also reveal a post-transcriptional regulatory machinery governing maturation from reticulocytes to erythrocytes.

Enhancing autophagy maturation with CCZ1-MON1A complex alleviates neuropathology and memory defects in Alzheimer disease models.

Rationale: Impairment of autophagy maturation has been implicated in Alzheimer's disease (AD) pathogenesis. However, the mechanism for this impairment has not been elucidated, and whether enhancing autophagy maturation is a viable therapeutic strategy for AD has not been verified.Methods: We examined the autophagosome maturation process in AD cell and mouse models by immunoblotting. To further understand the changes in autophagy in AD brains, we analyzed the transcriptome by RNA-sequencing and measured the expression of RAB7, CCZ1 and MON1A.We performed brain stereotaxic injections of AAV into 3xTg AD mouse brain and WT mouse brain to over-express MON1A/CCZ1 or knockdown MON1A. For in vitro studies, we purified autophagosomes, and determined GTP-RAB7 level in autophagosome fractions by GST-R7BD affinity-isolation assay.Results: We report that the active form of RAB7 was selectively decreased in autophagosome fractions isolated from cells and tissues of AD models, and that this decrease was accompanied by impaired activity of its guanine nucleotide exchange factor (GFE) CCZ1-MON1A. Overexpressing CCZ1-MON1A increased the active form of RAB7, enhanced autophagosome maturation, and promoted degradation of APP-CTFs, Aβ and P-tau in an autophagy-dependent manner in cells and a mouse AD model.Conclusions: Our data reveals that CCZ1-MON1A-RAB7 complex dysfunction is a potential mechanism for autophagosome maturation defects in AD, and advances the possibility that enhancing autophagosome maturation is a novel therapeutic strategy against AD.

Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward

BackgroundNewborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out. Materials/patients and methodsWe report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4–13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients. ResultsExtensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T). ConclusionThis study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening.

Early lysosome defects precede neurodegeneration with amyloid-β and tau aggregation in NHE6-null rat brain.

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-β levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-β and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-β. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-β and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-β and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-β and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-β and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

BOB.1/OBF.1 is required during B-cell ontogeny for B-cell differentiation and germinal center function.

BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia

Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rβ expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro, suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes.

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB1 alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia

AbstractX-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Abstract 12880: Neddylation Regulates Murine Cardiomyocyte Maturation

From birth to adulthood cardiomyocytes (CMs) undergo a complex maturation process characterized by drastic changes in cell size, ultrastructure, metabolism and gene expression. The molecular mechanisms regulating CM maturation remain poorly defined. Neddylation is a novel post-translational modification that covalently attaches a small ubiquitin-like protein, NEDD8, to target proteins. We found that deletion of NAE1, a critical NEDD8 E1 enzyme that triggers neddylation, in embryonic and perinatal mouse hearts via Nkx2.5 Cre and aMHC Cre , respectively, led to myocardial hypoplasia, heart failure and early lethality. The severe cardiac phenotypes are accompanied by disorganized sarcomere structure and defective fetal-to-adult gene transition, suggesting a possible role for neddylation in CM maturation. To rule out the confounding effect of secondary changes caused by perturbed CM differentiation and cardiac stress response in the mutant hearts, we used AAV9-cTnT-Cre to achieve mosaic knockout of NAE1 in postnatal hearts. High-dose AAV9 deleted NAE1 in 80% CMs and caused heart failure, whereas low-dose AVV9 deleted NAE1 in 30% CMs and preserved heart function. In the context of preserved cardiac function, CMs lacking NAE1 showed severe disrupted transverse tubules (T-tubules) network, which was further exacerbated in hearts with modest dysfunction. In contrast, T-tubule morphology was largely maintained in CMs with intact NAE1 expression from low-dose AAV9 infected hearts, as well as in CMs with deletion of one allele of NAE1, indicating a key, cell-autonomous role of neddylation in T-tubule maturation. Biochemical and histological analyses further revealed dysregulated metabolic and sarcomeric gene expression and disrupted sarcomere ultrastructure in high-dose AAV9 infected hearts, supporting defective CM maturation. In vitro, pharmacological inhibition of neddylation also impaired myofibril assembly in isolated neonatal and adult CMs. Together, our data reveal an important role of neddylation in CM maturation in the developing heart, warranting further investigations of detailed posttranslational mechanisms underlying CM maturation.

Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Purine Metabolism and Ribosomal Biogenesis during Myelopoiesis

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency.ResultsWe explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis.Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP.Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis.ConclusionOur studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. DisclosuresPorteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during “osteocytogenesis” is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Defective insulin maturation in patients with type 2 diabetes.

Progressive beta-cell dysfunction is a hallmark of type 2 diabetes (T2D). Increasing evidence indicates that over-stimulating proinsulin synthesis causes proinsulin misfolding and impairs insulin maturation and storage in db/db mice. However, defective insulin maturation in patients with T2D remains unknown. We examined intra-islet and intra-cellular distributions of proinsulin and insulin and proinsulin to insulin ratio in the islets of patients with T2D. The expression of transcription factor NKX6.1 and dedifferentiation marker ALDH1A3, as well as glucagon, were detected by immunofluorescence. We identified a novel subgroup of beta cells expressing only proinsulin but not insulin. Importantly, significantly increased proinsulin positive and insulin negative (PI+/INS-) cells were evident in T2D, and this increase was strongly correlated with levels of hemoglobin A1C (HbA1c) in T2D and prediabetes. The percentages of beta cells expressing prohormone convertase 1/3 and carboxypeptidase E were not reduced. Indeed, while proinsulin displayed a higher degree of co-localization with the golgi markers GM130/TGN46 in control beta cells, it appeared to be more diffused within the cytoplasm and less co-localized with GM130/TGN46 in PI+/INS- cells. Furthermore, the key functional transcription factor NKX6.1 markedly decreased in the islets of T2D, especially in the cells with PI+/INS-. The decreased NKX6.1+/PI+/INS+ was strongly correlated with levels of HbA1c in T2D. Almost all PI+/INS- cells showed absence of NKX6.1. Moreover, the percentages of PI+/INS- cells expressing ALDH1A3 were elevated along with an increased acquisition of glucagon immunostaining. Our data demonstrate defective insulin maturation in patients with T2D.

NATURAL KILLER CELLS PHENOTYPE CORRELATES WITH TYROSINE KINASE INHIBITORS TREATMENT RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS

Despite well-known Natural Killer (NK) cells antitumor activity, its potential in regulating Chronic Myeloid Leukemia (CML) is not fully stablished. Tyrosine kinase inhibitors (TKI) treatment, besides reducing BCR-ABL1 transcripts, seems to result in immunomodulation that partly restore the immune dysregulation of the disease. Numerical, functional and maturation defects might be associated with CML risk stratification, molecular response profile or even with resistance to treatment with TKI. Therefore, we aimed to immunophenotypically characterize frequency, subtypes and maturation profile of NK cells in CML patients, by Multiparametric Flow Cytometry (MFC), during monitoring of TKI treatment. 11 Peripheral Blood samples from patients with CML (Clinical Hospital of Ribeirão Preto - HCRP-USP) and 10 healthy donors (controls) were analyzed. Clinical, laboratory and ELN2017 risk stratification were assessed. NK cells were defined as CD19-CD3-CD56+ and: CD56brightCD16- (secretory), CD56dimCD16+ (cytotoxic), CD11b-CD27- (tolerant); CD27+CD11b- (immature secretor), CD27+CD11b+ (secretory) and CD11b+CD27- (cytotoxic). Based on molecular response levels assessed by BCR-ABL1 RT-qPCR, patients were classified in G1 (without loss of molecular response to the 1 st line of TKI; n = 3), G2 (loss of molecular response to 1 st line of TKI; n = 3) and G3 (loss of molecular response to 2 nd generation TKI; n = 5). Mann Whitney or Kruskal-Wallis tests (p < 0.05) were applied using SPSS (v.20). A higher frequency of lymphocytes in CML patients was observed when compared to controls (p = 0.0242), but no difference was found in the frequency of NK cells. G1 and G2 showed a higher frequency of NK cells than G3 (p = 0,7774). Even though no statistical significance was found, CD27-CD11b- and CD27+CD11b- NK cells showed a higher frequency, while CD27+CD11b+ NK cells showed a lower frequency in CML when compared to controls. Frequency of CD27+CD11b- NK cells was higher in G2 and G3 when compared to G1. G3 showed an immature profile with higher percentages of CD27-CD11b- and CD27+CD11b- NK cells when compared to controls, while the more mature CD27+CD11b+ NK cells showed a lower frequency in CML. Furthermore, a lower frequency of CD56brightCD16- (p = 0.0002) and CD56dimCD16+ (p = 0.0448) NK cells was observed in CML, as wells as the frequency of CD27-CD11b+ cytotoxic NK cells, consistent to what we found in G3 and supporting our hypothesis. We observed that patients with loss of molecular response in treatment with 1 st and 2 nd TKI generation presented an immature non-effective NK cell profile, with deficient cytotoxicity. This scenario might result in impaired tumor surveillance of CML, meaning that phenotypic changes in NK cells may directly affect risk stratification and treatment response in CML. However due to the small number of patients and the lack of a functional assay, it is not possible to confirm that the cytotoxic cell may actually be deficient. Our findings and further functional studies can contribute to a better understanding of the physiopathology, prognosis and monitoring of CML, response to TKI therapy, as well as help to define therapeutic strategies based on immunotherapy mediated by NK cells.