Gestational Diabetes Mellitus (GDM) is characterised by insulin resistance accompanied by reduced beta-cell compensation to increased insulin demand, typically observed in the second and third trimester and associated with adverse pregnancy outcomes. There is a need for a biomarker that can accurately monitor status and predict outcome in GDM, reducing foetal-maternal morbidity and mortality risks. To this end, circulating microRNAs (miRNAs) present themselves as promising candidates, stably expressed in serum and known to play crucial roles in regulation of glucose metabolism. We analysed circulating miRNA profiles in a cohort of GDM patients (n = 31) and nondiabetic controls (n = 29) during the third trimester for miRNA associated with insulin-secretory defects and glucose homeostasis. We identified miR-330-3p as being significantly upregulated in lean women with GDM compared to nondiabetic controls. Furthermore, increased levels of miR-330-3p were associated with better response to treatment (diet vs. insulin), with lower levels associated with exogenous insulin requirement. We observed miR-330-3p to be significantly related to the percentage of caesarean deliveries, with miR-330-3p expression significantly higher in spontaneously delivered GDM patients. We report this strong novel association of circulating miR-330-3p with risk of primary caesarean delivery as a pregnancy outcome linked with poor maternal glycaemic control, strengthening the growing body of evidence for roles of diabetes-associated miRNAs in glucose homeostasis and adaptation to the complex changes related to pregnancy. Gestational Diabetes Mellitus (GDM) is diagnosed as the development of hyperglycaemia during the second or third trimester in pregnant women not previously diagnosed with diabetes 1. The global epidemic of obesity and diabetes has resulted in an increase in the prevalence of diabetes mellitus in women of childbearing age and estimates currently place 5% to 20% of pregnancies as affected by GDM worldwide, due to differences in population demographics, diagnostic criteria, screening methods and maternal lifestyle 2. The significant risk-associations between hyperglycaemia not considered to be within the diagnostic range for overt diabetes and adverse perinatal and maternal outcomes require more accurate measures for monitoring status and predicting outcome in GDM 3. The pathophysiology of GDM manifests as a result of the progressive insulin resistance (IR) and subsequent increased insulin secretion requirement that occurs during normal pregnancy 4. As a result, most women with GDM develop pancreatic beta-cell dysfunction secondary to IR, resulting in impaired glucose tolerance in a setting of IR similar to that of T2DM 5. However, the possible causes of beta-cell dysfunction and IR are not well described, including the contributions of autoimmune and monogenic factors. Meta-analysis of gene expression profiles has indicated that the transcriptome profile in GDM is more similar to that of autoimmune type 1 diabetes mellitus (T1DM...
GGGGCC repeat expansion inC9orf72 is the most common genetic cause in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders in association with aging. Bidirectional repeat expansions in the noncoding region of C9orf72 have been shown to produce dipeptide repeat (DPR) proteins through repeat-associated non-ATG (RAN) translation and to reduce the expression level of the C9orf72 gene product, C9orf72 protein. Mechanisms underlying C9orf72-linked neurodegeneration include expanded RNA repeat gain of function, DPR toxicity, and C9orf72 protein loss of function. In the current study,we focus on the cellular function of C9orf72 protein. We report that C9orf72 can regulate lysosomal biogenesis and autophagy at the transcriptional level. We show that loss of C9orf72 leads to striking accumulation of lysosomes, autophagosomes, and autolysosomes in cells, which is associated with suppressed mTORC1 activity and enhanced nuclear translocation of MiT/TFE family members MITF, TFE3, and TFEB, three master regulators of lysosomal biogenesis and autophagy. We demonstrate that the DENN domain of C9orf72 specifically binds to inactive Rag GTPases, but not active Rag GTPases, thereby affecting the function of Rag/raptor/mTOR complex and mTORC1 activity. Furthermore, active Rag GTPases, but not inactive Rag GTPases or raptor rescued the impaired activity and lysosomal localization of mTORC1 in C9orf72-deficient cells. Taken together, the present study highlights a key role of C9orf72 in lysosomal and autophagosomal regulation, and demonstrates that Rag GTPases and mTORC1 are involved in C9orf72-mediated autophagy.
BackgroundChronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation.MethodsWe assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid−/− microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia.ResultsWe elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid−/− microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner.ConclusionsThis study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1143-3) contains supplementary material, which is available to authorized users.
Background Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
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