Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is ...restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.
Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with ...numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.
DIX-domain containing 1 (Dixdc1) is an important regulator of neuronal development including cortical neurogenesis, neuronal migration and synaptic connectivity, and sequence variants in the gene ...have been linked to autism spectrum disorders (ASDs). Previous studies indicate that Dixdc1 controls neurogenesis through Wnt signaling, whereas its regulation of dendrite and synapse development requires Wnt and cytoskeletal signaling. However, the prediction of these signaling pathways is primarily based on the structure of Dixdc1. Given the role of Dixdc1 in neural development and brain disorders, we hypothesized that Dixdc1 may regulate additional signaling pathways in the brain. We performed transcriptomic and proteomic analyses of Dixdc1 KO mouse cortices to reveal such alterations. We found that transcriptomic approaches do not yield any novel findings about the downstream impacts of Dixdc1. In comparison, our proteomic approach reveals that several important mitochondrial proteins are significantly dysregulated in the absence of Dixdc1, suggesting a novel function of Dixdc1.
The development of neural connectivity is essential for brain function, and disruption of this process is associated with autism spectrum disorders (ASDs). DIX domain containing 1 (DIXDC1) has ...previously been implicated in neurodevelopmental disorders, but its role in postnatal brain function remains unknown. Using a knockout mouse model, we determined that DIXDC1 is a regulator of excitatory neuron dendrite development and synapse function in the cortex. We discovered that MARK1, previously linked to ASDs, phosphorylates DIXDC1 to regulate dendrite and spine development through modulation of the cytoskeletal network in an isoform-specific manner. Finally, rare missense variants in DIXDC1 were identified in ASD patient cohorts via genetic sequencing. Interestingly, the variants inhibit DIXDC1 isoform 1 phosphorylation, causing impairment to dendrite and spine growth. These data reveal that DIXDC1 is a regulator of cortical dendrite and synaptic development and provide mechanistic insight into morphological defects associated with neurodevelopmental disorders.
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•DIXDC1 is a regulator of dendrite and spine development•MARK1 phosphorylates DIXDC1 isoforms to regulate dendrite and spine development•Phosphorylation of DIXDC1 isoform 1 regulates cytoskeletal dynamics•ASD variants in DIXDC1 isoform 1 impair phosphorylation and neuronal morphology
Dendritic and synaptic development are important for brain function, and these events are disrupted in individuals with neurodevelopmental disorders. Kwan et al. show that MARK1-dependent phosphorylation of DIXDC1 regulates cortical neuron dendrite and spine morphogenesis through a cytoskeletal pathway that is disrupted by rare genetic variants identified in autism cohorts.
Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and are known to play a central role in the growth and ...differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFβ superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined.
Accordingly, GDF10−/− mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10.
Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10−/− mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10−/− mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis.
Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury.
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•The activation of PPARγ in hepatocytes is antagonized by GDF10.•GDF10 attenuates lipid accumulation in cultured hepatocytes.•GDF10−/− mice develop diet-induced obesity and severe hepatic steatosis.•HFD-fed GDF10−/− mice exhibit hepatic ER stress, fibrosis and injury.•GDF10 is a novel hepatoprotective member of the TGFβ superfamily.
The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function ...(LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78–PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.
The proprotein convertase subtilisin/kexin type‐9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low‐density lipoprotein receptor (LDLR). As such, loss‐of‐function ...(LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR); in this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis were not observed. Further, ER‐retention of endogenous PCSK9 via splice‐switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94‐kDa glucose regulated protein (GRP94) sequesters PCSK9 away from the 78‐kDa glucose regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78‐PCSK9 complex and resulted in UPR activation following overexpression of ER‐retained PCSK9 variants relative to wild‐type secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.
Support or Funding Information
This work was supported in part by research grants to Richard C. Austin from the Heart and Stroke Foundation of Ontario (T‐6146), the Heart and Stroke Foundation of Canada (G‐13‐0003064 and G‐15‐0009389), the Canadian Institute of Health Research (74477)
Unfolded protein response sensor GRP78 detects misfolded protein in the ER, such as ER‐retained vasopressin mutant VPG17V, dissociates from UPR transducers ATF6, IRE1α and PERK thereby leading to UPR activation. In contrast, Misfolded PCSK9 mutants, such as PCSK9Q152H, are masked from GRP78 by ER‐resident chaperone GRP94. This study highlights a novel mechanism by which certain misfolded proteins, arising from heritable mutations, fail to cause ER stress and UPR activation.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Cholangiocarcinoma (CCA) is a molecularly heterogenous disease that is often fatal. Whole genome sequencing (WGS) can provide additional knowledge of mutational spectra compared with panel ...sequencing. We describe the molecular landscape of CCA using whole-genome sequencing and compare the mutational landscape between short-term and long-term survivors.
We explored molecular differences between short-term and long-term survivors by performing WGS on 20 patient samples from our biliary tract cancer database. Short-term survivors were enriched for cases with underlying primary sclerosing cholangitis (PSC) and patients with cirrhosis. All samples underwent tumour epithelial enrichment using laser capture microdissection (LCM).
Dominant single base substitution (SBS) signatures across the cohort included SBS1 and SBS5, with the latter more prevalent in long-term survivors. SBS17 was evident in 3 cases, all of whom had underlying ulcerative colitis (UC) with PSC. Additional rare signatures included SBS3 in a patient treated for prior mantle cell lymphoma and SBS26/SBS6 in a patient with a tumor mutational burden of 33 mutations/Mb and a pathogenic
germline mutation. Somatic
inactivating mutations were present in 8/10 (80%) short-term survivors and in none of the long-term survivors. Additional mutations occurred in
, and chromatin remodelling genes. The long-term survivor group harboured predicted fusions in
(n=2) and pathogenic mutations in
and
(n=2).
alterations are associated with poor outcomes in patients with CCA. Patients with underlying inflammatory/autoimmune conditions may be enriched for unique tumour mutational signatures.
With the emergence of the cancer stem cell hypothesis in leukemia there has been a need to develop the crucial tools and assays to prospectively isolate these cells from other cancers. This review ...focuses on the latest strategies to prospectively isolate cancer stem cells and also explores some of the caveats of the methodology that has taken hold. Emerging themes in the cancer stem cell field will be explored, including relevance of cell of origin, intraclonal heterogeneity, and how exploiting the unique functional attributes of stemness in the cancer stem cell population can refine approaches to isolate these cells from various malignancies.
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