SummaryOur study identifies a transcriptional role of cell death-inducing DNA fragmentation factor-like effector A (CIDEA), a lipid-droplet-associated protein, whereby it regulates human adipocyte britening/beiging with consequences for the regulation of energy expenditure. The comprehensive transcriptome analysis revealed CIDEA's control over thermogenic function in brite/beige human adipocytes. In the absence of CIDEA, achieved by the modified dual-RNA-based CRISPR-Cas9nD10A system, adipocytes lost their britening capability, which was recovered upon CIDEA re-expression. Uncoupling protein 1 (UCP1), the most upregulated gene in brite human adipocytes, was suppressed in CIDEA knockout (KO) primary human adipocytes. Mechanistically, during induced britening, CIDEA shuttled from lipid droplets to the nucleus via an unusual nuclear bipartite signal in a concentration-dependent manner. In the nucleus, it specifically inhibited LXRα repression of UCP1 enhancer activity and strengthened PPARγ binding to UCP1 enhancer, hence driving UCP1 transcription. Overall, our study defines the role of CIDEA in increasing thermogenesis in human adipocytes.

White adipocytes are specialized cells that have a very high capacity to esterify and store FAs as triglyceride (TAG) within a single lipid droplet (LD) and release them as required by variations in nutritional state (1). In obesity, the adipocyte must cope with an increased flux of FAs from the diet and higher basal lipolysis (2, 3). If these FAs are not efficiently esterified or oxidized, unbound FAs within the cell cause lipotoxic stress with deleterious consequences for adipocyte metabolic and endocrine function (4-6). As the storage capacity of the adipocyte is exceeded in obesity, the overflow of FAs can lead to ectopic fat deposition and systemic metabolic dysfunction (1,7,8). Strategies to modify the metabolism of FAs in adipocytes have therapeutic potential in obesity-related metabolic diseases.In contrast to multilocular brown or "brite"/beige adipocytes that are densely packed with mitochondria and express UCP1, white adipocytes contain relatively few mitochondria, low FA oxidation capacity, and much lower levels of UCP1 (9). White adipose tissue (WAT), however, can acquire brown-like features in response to -adrenergic stimulation or cold. Studies in rodents have shown that brite/beige adipocytes can be formed from mature white adipocytes (10, 11) or the recruitment and differentiation of beige progenitors (12,13). Several lines of evidence indicate that mature white human adipocytes can be metabolically reprogrammed to a more oxidative phenotype in Abstract Treatment with PPAR agonists in vivo improves human adipocyte metabolism, but the cellular mechanisms and possible depot differences in responsiveness to their effects are poorly understood. To examine the ex vivo metabolic effects of rosiglitazone (Rosi), we cultured explants of human visceral (omental) and abdominal subcutaneous adipose tissues for 7 days. Rosi increased mRNA levels of transcriptional regulators of brite/beige adipocytes (PGC1, PRDM16), triglyceride synthesis (GPAT3, DGAT1), and lipolysis (ATGL) similarly in adipose tissues from both depots. In parallel, Rosi increased key modulators of FA oxidation (UCP1, FABP3, PLIN5 protein), rates of FA oxidation, and protein levels of electron transport complexes, suggesting an enhanced respiratory capacity as confirmed in newly differentiated adipocytes. Rosi led to the formation of small lipid droplets (SLDs) around the adipocyte central lipid droplet; each SLD was decorated with redistributed mitochondria that colocalized with PLIN5. SLD maintenance required lipolysis and FA reesterification. Rosi thus coordinated a structural and metabolic remodeling in adipocytes from both visceral and subcutaneous depots that enhanced oxidative capacity. Selective targeting of these cellular mechanisms to improve adipocyte FA handling may provide a new approach to treat metabolic complications of obesity and diabetes.-Lee, M-J., S. Jash, J. E. C. Jones, V. Puri, and S. K. Fried. Rosiglitazone remodels the lipid droplet and britens human visceral and subcutaneous adipocytes ex vivo. J. Lipid Res. 20...

A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimer’s disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n = 33 early onset and 33 late onset) and gestational age-matched controls (n = 77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n = 24) stages with age-matched controls (n = 19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid β-42, α-synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949–1.00; AD; AUC: 0.986, CI: 0.832–1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE.

Skeletal muscle injury is associated with general down-regulation of mitochondrial function. Postinjury regeneration of skeletal muscle occurs through activation, proliferation, and differentiation of resident stem cells, including satellite cells and endothelial precursor cells. We wanted to determine the role of mitochondrial function in the regeneration process. Using a previously described method for complex-mediated delivery to intracellular mitochondria, a combination of polycistronic RNAs encoding the H strand of the rat mitochondrial genome was administered to injured rat quadriceps muscle, resulting in restoration of mitochondrial mRNA levels, organellar translation, and respiratory capacity. Intramuscular ATP levels were elevated on pcRNA treatment of injured muscle; concomitantly, levels of reactive oxygen species in the injured muscle were reduced. These effects combined to produce a notable increase in the rate of wound resolution, accompanied by reduction of fibrosis and acceleration of myogenesis, vasculogenesis, and resumption of muscle contractile function. There was evidence of proliferation of Pax7+ satellite cells, expression of muscle-specific regulatory factors in a specific time sequence, and formation of new myofibers in the regenerating muscle. RNA-induced wound resolution and satellite cell proliferation were sensitive to mitochondrial inhibitors, indicating the importance of oxidative phosphorylation. These results highlight the activation of endogenous stem cells through mitochondrial restoration as a possible alternative to implantation of cultured stem cells.

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Pathogens comprised of viruses, bacteria, gut microbiome, and parasites are a leading cause of ever-emerging diseases in humans. Studying pathogens for their ability to cause diseases is a topic of critical discussion among scientists and pharmaceutical centers for effective drug development that diagnose, treat, and prevent infection-associated disorders. Pathogens impact health either directly by invading the host or by eliciting an acute inflammatory immune response. This paradigm of inflammatory immune responses is even more consequential in people who may be immunocompromised. In this regard, pregnancy offers an altered immunity scenario, which may allow the onset of severe diseases. Viruses, such as Influenza, HIV, and now SARS-CoV-2, associated with the COVID-19 pandemic, raise new concerns for maternal and fetal/neonatal health. Intrauterine bacterial and parasitic infections are also known to impact pregnancy outcomes and neonatal health. More importantly, viral and bacterial infections during pregnancy have been identified as a common contributor to fetal brain development defects. Infection-mediated inflammatory uterine immune milieu is thought to be the main trigger for causing poor fetal brain development, resulting in long-term cognitive impairments. The concept of in utero programming of childhood and adult disorders has revolutionized the field of neurodevelopment and its associated complications. Recent findings in mice and humans clearly support the idea that uterine immunity during pregnancy controls the health trajectory of the child and considerably influences the cognitive function and mental health. In this review, we focus on the in utero programming of autism spectrum disorders (ASD) and assess the effects of pathogens on the onset of ASD-like symptoms.

The microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to short-term local hypoxia, but there are conflicting reports on the role of hypoxia on the myogenic program in vivo and in vitro. In our rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded RNAs results in satellite cell proliferation and initiation of myogenesis. We now report that MR leads to a transient hypoxic response in situ. Inhibition of hypoxia by lowering mitochondrial O2 consumption, either by respiratory electron transport inhibitors, or by NO-mediated inhibition of O2 binding to cytochrome c oxidase, resulted in exacerbation of inflammation. Lentivirus-mediated knockdown of hypoxia-inducible factor 1α (HIF1α) or of Notch signaling components had a similar effect, and pharmacologic inhibition of HIF or Notch reduced the number of proliferating Pax7+ cells. In contrast, a prolonged hypoxic response induced either by uncoupling of respiration from oxidative phosphorylation or through HIF stabilization by dimethyloxalylglycine (DMOG) had an immediate anti-inflammatory effect. Although significant satellite cell proliferation occurred in presence of DMOG, expression of differentiation markers was affected. These results emphasize the importance of transient hypoxia as opposed to prolonged hypoxia for myogenesis.

During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3= untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis. R egeneration of skeletal muscle following injury involves formation of new myofibers as well as patch repair of old injured fibers by fusion of satellite cell-derived myoblasts (1). The program of differentiation of satellite cells involves sequential expression of myogenic regulatory factors (MRFs) and their target genes (2-5). Mitochondrial biogenesis occurs at a specific time prior to the formation of myofibers (4, 6), suggesting an energy-requiring step, but the specific role of ATP in differentiation remains unknown. Eukaryotic cells contain a number of enzymes that sense the energy status, particularly, the pools of AMP and ADP (AMPactivated protein kinase [AMPK]) or of ATP (mammalian Target of Rapamycin [mTOR] kinases) (7,8). It is possible that mitochondrial activity, through modulation of the adenylate pool, impacts these energy sensors, thereby activating or inhibiting downstream pathways.Although energy sensors such as AMPK and mTORC are known to regulate cellular energy homeostasis and cell growth (9), there are indications that they could also modulate specific developmental processes. It was shown early that the mTORC inhibitor rapamycin inhibits growth of myofibers in regenerating adult muscle (10); such inhibition was overcome by expression of a rapamycin-resistant mTORC gene (11). Genetic ablation of mTORC (12), or of the mTORC1 subunit Raptor (13), or of the mTORC1 target S6K (14) results in severe muscle atrophy. These studies indicate that myofiber growth and maturation are regulated by mTORC1 acting through S6K in vivo but provide no information on the specific eff...