Long Noncoding RNA VLDLR-AS1 Levels in Serum Correlate with Combat-Related Chronic Mild Traumatic Brain Injury and Depression Symptoms in US Veterans.

More than 75% of traumatic brain injuries (TBIs) are mild (mTBI) and military service members often experience repeated combat-related mTBI. The chronic comorbidities concomitant with repetitive mTBI (rmTBI) include depression, post-traumatic stress disorder or neurological dysfunction. This study sought to determine a long noncoding RNA (lncRNA) expression signature in serum samples that correlated with rmTBI years after the incidences. Serum samples were obtained from Long-Term Impact of Military-Relevant Brain-Injury Consortium Chronic Effects of Neurotrauma Consortium (LIMBIC CENC) repository, from participants unexposed to TBI or who had rmTBI. Four lncRNAs were identified as consistently present in all samples, as detected via droplet digital PCR and packaged in exosomes enriched for CNS origin. The results, using qPCR, demonstrated that the lncRNA VLDLR-AS1 levels were significantly lower among individuals with rmTBI compared to those with no lifetime TBI. ROC analysis determined an AUC of 0.74 (95% CI: 0.6124 to 0.8741; p = 0.0012). The optimal cutoff for VLDLR-AS1 was ≤153.8 ng. A secondary analysis of clinical data from LIMBIC CENC was conducted to evaluate the psychological symptom burden, and the results show that lncRNAs VLDLR-AS1 and MALAT1 are correlated with symptoms of depression. In conclusion, lncRNA VLDLR-AS1 may serve as a blood biomarker for identifying chronic rmTBI and depression in patients.

Female and male obese Zucker rats display differential inflammatory mediator and long non-coding RNA profiles.

AIMS: The goal of this study was to identify mediators in peri-lymphatic adipose tissue (PLAT) that are altered in obese versus lean Zucker rats, with focus on potential sex differences MAIN METHODS: Mesenteric PLAT was analyzed with protein and lncRNA arrays. Additional RT-PCR confirmation was performed with epididymal/ovarian fat. KEY FINDINGS: MCP-1, TCK-1, Galectin-1, Galectin-3, and neuropilin-1 were elevated in PLAT from obese rats of both sexes. However, 11 additional proteins were elevated only in obese males while 24 different proteins were elevated in obese females. Profiling of lncRNAs revealed lean males have elevated levels of NEAT1, MALAT1 and GAS5 compared to lean females. NEAT1, MALAT1, and GAS5 were significantly reduced with obesity in males but not in females. Another lncRNA, HOTAIR, was higher in lean females compared to males, and its levels in females were reduced with obesity. Obese rats of both sexes had similar histologic findings of mesenteric macrophage crown-like structures and hepatocyte fat accumulation. SIGNIFICANCE: While obese male and female Zucker rats both have increased inflammation, they have distinct signals. Future studies of the proteome and lncRNA landscape of obese males vs. females in various animal models and in human subjects are warranted to better guide development of therapeutics for obesity-induced inflammation.

Regulation of Human Sortilin Alternative Splicing by Glucagon-like Peptide-1 (GLP1) in Adipocytes.

Type 2 diabetes mellitus is a chronic metabolic disease with no cure. Adipose tissue is a major site of systemic insulin resistance. Sortilin is a central component of the glucose transporter -Glut4 storage vesicles (GSV) which translocate to the plasma membrane to uptake glucose from circulation. Here, using human adipocytes we demonstrate the presence of the alternatively spliced, truncated sortilin variant (Sort_T) whose expression is significantly increased in diabetic adipose tissue. Artificial-intelligence-based modeling, molecular dynamics, intrinsically disordered region analysis, and co-immunoprecipitation demonstrated association of Sort_T with Glut4 and decreased glucose uptake in adipocytes. The results show that glucagon-like peptide-1 (GLP1) hormone decreases Sort_T. We deciphered the molecular mechanism underlying GLP1 regulation of alternative splicing of human sortilin. Using splicing minigenes and RNA-immunoprecipitation assays, the results show that GLP1 regulates Sort_T alternative splicing via the splice factor, TRA2B. We demonstrate that targeted antisense oligonucleotide morpholinos reduces Sort_T levels and improves glucose uptake in diabetic adipocytes. Thus, we demonstrate that GLP1 regulates alternative splicing of sortilin in human diabetic adipocytes.

Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function.

BACKGROUND: The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it's expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. METHODS: ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). RESULTS: Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. CONCLUSIONS: Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. Video Abstract.

Small molecule targeting long noncoding RNA GAS5 administered intranasally improves neuronal insulin signaling and decreases neuroinflammation in an aged mouse model.

Shifts in normal aging set stage for neurodegeneration and dementia affecting 1 in 10 adults. The study demonstrates that lncRNA GAS5 is decreased in aged and Alzheimer's disease brain. The role and targets of lncRNA GAS5 in the aging brain were elucidated using a GAS5-targeting small molecule NPC86, a frontier in lncRNA-targeting therapeutic. Robust techniques such as molecular dynamics simulation of NPC86 binding to GAS5, in vitro functional assays demonstrating that GAS5 regulates insulin signaling, neuronal survival, phosphorylation of tau, and neuroinflammation via toll-like receptors support the role of GAS5 in maintaining healthy neurons. The study demonstrates the safety and efficacy of intranasal NPC86 treatment in aged mice to improve cellular functions with transcriptomic analysis in response to NPC86. In summary, the study demonstrates that GAS5 contributes to pathways associated with neurodegeneration and NPC86 has tremendous therapeutic potential to prevent the advent of neurodegenerative diseases and dementias.

Induction of beige-like adipocyte markers and functions in 3T3-L1 cells by Clk1 and PKCßII inhibitory molecules.

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre-mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre-mRNA. Clk1 inhibition by TG003 results in beige-like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003-treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCßII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCßII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.

Role of alternatively spliced, pro-survival Protein Kinase C delta VIII (PKCδVIII) in ovarian cancer.

Ovarian cancer is the deadliest malignant disease in women. Protein Kinase C delta (PRKCD; PKCδ) is serine/threonine kinase extensively linked to various cancers. In humans, PKCδ is alternatively spliced to PKCδI and PKCδVIII. However, the specific function of PKCδ splice variants in ovarian cancer has not been elucidated yet. Hence, we evaluated their expression in human ovarian cancer cell lines (OCC): SKOV3 and TOV112D, along with the normal T80 ovarian cells. Our results demonstrate a marked increase in PKCδVIII in OCC compared to normal ovarian cells. Therefore, we elucidated the role of PKCδVIII and the underlying mechanism of its expression in OCC. Using overexpression and knockdown studies, we demonstrate that PKCδVIII increases cellular survival and migration in OCC. Further, overexpression of PKCδVIII in T80 cells resulted in increased expression of Bcl2 and knockdown of PKCδVIII in OCC decreased Bcl2 expression. Using co-immunoprecipitations and immunocytochemistry, we demonstrate nuclear localization of PKCδVIII in OCC and further show increased association of PKCδVIII with Bcl2 and Bcl-xL in OCC. Using PKCδ splicing minigene, mutagenesis, siRNA and antisense oligonucleotides, we demonstrate that increased levels of alternatively spliced PKCδVIII in OCC is regulated by splice factor SRSF2. Finally, we verified that PKCδVIII levels are elevated in samples of human ovarian cancer tissue. The data presented here demonstrate that the alternatively spliced, signaling kinase PKCδVIII is a viable target to develop therapeutics to combat progression of ovarian cancer.

Long Noncoding RNA GAS5 Contained in Exosomes Derived from Human Adipose Stem Cells Promotes Repair and Modulates Inflammation in a Chronic Dermal Wound Healing Model.

Chronic recalcitrant wounds result from delayed or slowed healing processes. Underlying inflammation is a substantial risk factor for impaired dermal wound healing and often leads to chronic wound-related sequelae. Human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The goal of this project was to improve the outcome of chronic wounds by harvesting the exosomes from hASCs for therapeutic intervention. The results demonstrate that long noncoding RNA GAS5 is highly enriched in hASC exosomes and, further, that GAS5 is central to promoting wound repair in vitro. To evaluate the outcome of wound healing in a chronic low-grade inflammatory environment, lipopolysaccharide-treated HDF cells were evaluated for their response to hASC exosome treatment. Ingenuity pathway analysis identified inflammation pathways and genes affected by exosomes in a GAS5-dependent manner. Using siRNA to deplete GAS5 in HDF, the results demonstrated that Toll-like receptor 7 (TLR7) expression levels were regulated by GAS5. Importantly, the results demonstrate that GAS5 regulates inflammatory pathway genes in a chronic inflammation environment. The results presented here demonstrate that hASC exosomes are a viable therapeutic that accelerate the healing of chronic recalcitrant wounds.

Intranasal delivery of exosomes from human adipose derived stem cells at forty-eight hours post injury reduces motor and cognitive impairments following traumatic brain injury.

The neuroprotective role of human adipose-derived stems cells (hASCs) has raised great interest in regenerative medicine due to their ability to modulate their surrounding environment. Our group has demonstrated that exosomes derived from hASC (hASCexo) are a cell-free regenerative approach to long term recovery following traumatic brain injury (TBI). Previously, we demonstrated the efficacy of exosome treatment with intravenous delivery at 3 h post TBI in rats. Here, we show efficacy of exosomes through intranasal delivery at 48 h post TBI in mice lengthening the therapeutic window of treatment and therefore increasing possible translation to clinical studies. Our findings demonstrate significant recovery of motor impairment assessed by an elevated body swing test in mice treated with exosomes containing MALAT1 compared to both TBI mice without exosomes and exosomes depleted of MALAT1. Significant cognitive improvement was seen in the reversal trial of 8 arm radial arm water maze in mice treated with exosomes containing MALAT1. Furthermore, cortical damage was significantly reduced in mice treated with exosomes containing MALAT1 as well as decreased MHCII+ staining of microglial cells. Mice without exosomes or treated with exosomes depleted of MALAT1 did not show similar recovery. Results demonstrate both inflammation related genes and NRTK3 (TrkC) are target genes modulated by hASC exosomes and further that MALAT1 in hASC exosomes regulates expression of full length TrkC thereby activating the MAPK pathway and promoting recovery. Exosomes are a promising therapeutic approach following TBI with a therapeutic window of at least 48 h and contain long noncoding RNA's, specifically MALAT1 that play a vital role in the mechanism of action.

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes.

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein-protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.

Multilevel Regulation of Protein Kinase CδI Alternative Splicing by Lithium Chloride.

Lithium chloride (LiCl) is commonly used in treatment of mood disorders; however, its usage leads to weight gain, which promotes metabolic disorders. Protein kinase C delta (PKCδ), a serine/threonine kinase, is alternatively spliced to PKCδI and PKCδII in 3T3-L1 cells. We previously demonstrated that PKCδI is the predominantly expressed isoform in 3T3-L1 preadipocytes. Here, we demonstrate that LiCl treatment decreases PKCδI levels, increases formation of lipid droplets, and increases oxidative stress. Hence, we investigated the molecular mechanisms underlying the regulation of PKCδI alternative splicing by LiCl. We previously demonstrated that the splice factor SFRS10 is essential for PKCδI splicing. Our results demonstrate that glycogen synthase kinase 3 beta (GSK3ß) phosphorylates SFRS10, and SFRS10 is in a complex with long noncoding RNA NEAT1 to promote PKCδI splicing. Using PKCδ splicing minigene and RNA immunoprecipitation assays, our results demonstrate that upon LiCl treatment, NEAT1 levels are reduced, GSK3ß activity is inhibited, and SFRS10 phosphorylation is decreased, which leads to decreased expression of PKCδI. Integration of the GSK3ß signaling pathway with the ribonucleoprotein complex of long noncoding RNA (lncRNA) NEAT1 and SFRS10 enables fine-tuning of PKCδI expression during adipogenesis. Knowledge of the molecular pathways impacted by LiCl provides an understanding of the ascent of obesity as a comorbidity in disease management.

A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals.

The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKCδI is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKCδI activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKCδI isozymes and a caspase-3 binding site on the PKCδI hinge domain with Schrödinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKCδI. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKCδI and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKCδI, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKCδI activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKCδI catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKCδI catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKCδI is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3-mediated activation of kinase activity.

Stabilization of lncRNA GAS5 by a Small Molecule and Its Implications in Diabetic Adipocytes.

Long noncoding RNA (lncRNA) are regulatory RNAs >200 nt. We previously showed that lncRNA GAS5 decreases significantly in serum of type 2 diabetes mellitus (T2DM) patients. Hence, we sought to decipher the molecular mechanisms underlying the role of GAS5 in T2DM in adipose tissue. Using CHIP-RIP, we demonstrate that GAS5 binds to promoter of insulin receptor to regulate its expression, and its depletion inhibits glucose uptake and insulin signaling. Toward stabilizing GAS5 levels in T2DM, we incorporated a strategy to limit the degradation of GAS5 by blocking the interaction of GAS5 and UPF1 with a small molecule identified using OBTC screening strategy. NP-C86 binds to GAS5 with high affinity, and increases GAS5 levels and glucose uptake in diabetic patient adipocytes. As a broader impact, NP-C86 may be used as a molecular probe to investigate the intricacies of GAS5 in relevant biological systems as it offers specificity, efficient cellular uptake and is non-cytotoxic.

Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing.

Objective: Chronically ill patients heal recalcitrant ulcerative wounds more slowly. Human adipose-derived stem cells (hADSCs) play an important role in tissue regeneration and exosomes secreted by hADSC contribute to their paracrine signaling. In addition to cytokines, lipids and growth factors, hADSC secrete mRNA, miRNA, and long noncoding (lnc) RNA into exosomes. In this study we examined the role of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), an abundant lncRNA in exosomes from conditioned media (CM), on cell migration and ischemic wound healing. Approach: CM and isolated exosomes from hADSC were applied to human dermal fibroblast (HDF) in scratch assays and electric cell-substrate impedance sensing (ECIS) assays. CM was also applied to a rat model of ischemic wound healing and wound closure was followed. Results: CM stimulated cell migration of HDFs in vitro by 48%. CM stimulated the closure of ischemic wounds in a rat model 50% faster than unconditioned media. The depletion of MALAT1 in adipose-derived stem cell (ADSC) CM significantly reduced cell migration. Since MALAT1 is secreted into exosomes, a purified population of exosomes was applied to HDF where they enhanced cell migration in a similar manner to FGF-2 or basic fibroblast growth factor (bFGF) in ECIS wound healing assays. The uptake of exosomes by HDF was shown using dynasore, an inhibitor that blocks clathrin- and caveolin-dependent endocytosis. Depletion of MALAT1 in hADSC with antisense oligonucleotides resulted in exosomes without MALAT1. These exosomes had an effect similar to the unconditioned, control media in ECIS assays. Innovation: Exosomes contain lncRNA MALAT1 and other factors that have the potential to stimulate HDF cell migration and angiogenesis involved in wound healing without applying stem cells to wounds. Conclusion: Our results show the potential of using topically applied ADSC-derived exosomes containing MALAT1 for treating ischemic wounds. This allows for harnessing the power of stem cell paracrine signaling capabilities without applying the cells.

Long noncoding RNA MALAT1 in exosomes drives regenerative function and modulates inflammation-linked networks following traumatic brain injury.

BACKGROUND: Neuroinflammation is a common therapeutic target for traumatic brain injury (TBI) due to its contribution to delayed secondary cell death and has the potential to occur for years after the initial insult. Exosomes from adipose-derived stem cells (hASCs) containing the long noncoding RNA MALAT1 are a novel, cell-free regenerative approach to long-term recovery after traumatic brain injury (TBI) that have the potential to modulate inflammation at the genomic level. The long noncoding RNA MALAT1 has been shown to be an important component of the secretome of hASCs. METHODS: We isolated exosomes from hASC containing or depleted of MALAT1. The hASC-derived exosomes were then administered intravenously to rats following a mild controlled cortical impact (CCI). We followed the rats with behavior, in vivo imaging, histology, and RNA sequencing (RNA Seq). RESULTS: Using in vivo imaging, we show that exosomes migrate into the spleen within 1 h following administration and enter the brain several hours later following TBI. Significant recovery of function on motor behavior as well as a reduction in cortical brain injury was observed after TBI in rats treated with exosomes. Treatment with either exosomes depleted of MALAT1 or conditioned media depleted of exosomes showed limited regenerative effects, demonstrating the importance of MALAT1 in exosome-mediated recovery. Analysis of the brain and spleen transcriptome using RNA Seq showed MALAT1-dependent modulation of inflammation-related pathways, cell cycle, cell death, and regenerative molecular pathways. Importantly, our data demonstrates that MALAT1 regulates expression of other noncoding RNAs including snoRNAs. CONCLUSION: We demonstrate that MALAT1 in hASC-derived exosomes modulates multiple therapeutic targets, including inflammation, and has tremendous therapeutic potential for treatment of TBI.

MALAT1 in Human Adipose Stem Cells Modulates Survival and Alternative Splicing of PKCδII in HT22 Cells.

Brain injury may be caused by trauma or may occur in stroke and neurodegenerative diseases. Because the central nervous system is unable to regenerate efficiently, there is utmost interest in the use of stem cells to promote neuronal survival. Of interest here are human adipose-derived stem cells (hASCs), which secrete factors that enhance regeneration and survival of neurons in sites of injury. We evaluated the effect of hASC secretome on immortalized mouse hippocampal cell line (HT22) after injury. Protein kinase C δ (PKCδ) activates survival and proliferation in neurons and is implicated in memory. We previously showed that alternatively spliced PKCδII enhances neuronal survival via B-cell lymphoma 2 Bcl2 in HT22 neuronal cells. Our results demonstrate that following injury, treatment with exosomes from the hASC secretome increases expression of PKCδII in HT22 cells and increases neuronal survival and proliferation. Specifically, we demonstrate that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA contained in the hASC exosomes mediates PKCδII splicing, thereby increasing neuronal survival. Using antisense oligonucleotides for MALAT1 and RNA immunoprecipitation assays, we demonstrate that MALAT1 recruits splice factor serine-arginine-rich splice factor 2 (SRSF2) to promote alternative splicing of PKCδII. Finally, we evaluated the role of insulin in enhancing hASC-mediated neuronal survival and demonstrated that insulin treatment dramatically increases the association of MALAT1 and SRSF2 and substantially increases survival and proliferation after injury in HT22 cells. In conclusion, we demonstrate the mechanism of action of hASC exosomes in increasing neuronal survival. This effect of hASC exosomes to promote wound healing can be further enhanced by insulin treatment in HT22 cells.

Adipose-derived stem cells from lean and obese humans show depot specific differences in their stem cell markers, exosome contents and senescence: role of protein kinase C delta (PKCδ) in adipose stem cell niche.

BACKGROUND: Adipose-derived stem cells (ASC) and its exosomes are gaining utmost importance in the field of regenerative medicine. The ASCs tested for their potential in wound healing are predominantly derived from the subcutaneous depot of lean donors. However, it is important to characterize the ASC derived from different adipose depots as these depots have clinically distinct roles. METHODS: We characterized the ASC derived from subcutaneous and omental depots from a lean donor (sc-ASCn and om-ASCn) and compared it to the ASC derived from an obese donor (sc-ASCo and om-ASCo) using flow cytometry and real time qPCR. RESULTS: We show that stem cell markers Oct4, Sal4, Sox15, KLF4 and BMI1 have distinct expression patterns in each ASC. We evaluated the secretome of the ASC and characterized their secreted exosomes. We show long noncoding RNAs (lncRNAs) are secreted by ASC and their expression varied between the ASC's derived from different depots. Protein kinase C delta (PKCδ) regulates the mitogenic signals in stem cells. We evaluated the effect of silencing PKCδ in sc-ASCn, om-ASCn, sc-ASCo and om-ASCo. Using ß-galactosidase staining, we evaluated the percentage of senescent cells in sc-ASCn, om-ASCn, sc-ASCo and om-ASCo. Our results also indicated that silencing PKCδ increases the percentage of senescent cells. CONCLUSIONS: Our case-specific study demonstrates a role of PKCδ in maintaining the adipose stem cell niche and importantly demonstrates depot-specific differences in adipose stem cells and their exosome content.

Circulating long noncoding RNA GAS5 levels are correlated to prevalence of type 2 diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM), a metabolic disease, is characterized by impaired fasting glucose levels. Type 2 DM is adult onset diabetes. Long non-coding RNAs (lncRNAs) regulate gene expression and multiple studies have linked lncRNAs to human diseases. METHODS: Serum samples obtained from 96 participating veterans at JAH VA were deposited in the Research Biospecimen Repository. We used a two-stage strategy to identify an lncRNA whose levels correlated with T2DM. Initially we screened five serum samples from diabetic and non-diabetic individuals using lncRNA arrays. Next, GAS5 lncRNA levels were analyzed in 96 serum samples using quantitative PCR. Receiver operating characteristic (ROC) analysis was performed to determine the optimal cutoff GAS5 for diagnosis of DM. RESULTS: Our results demonstrate that decreased GAS5 levels in serum were associated with diabetes in a cohort of US military veterans. The ROC analysis revealed an optimal cutoff GAS5 value of less than or equal to 10. qPCR results indicated that individuals with absolute GAS5 < 10 ng/µl have almost twelve times higher odds of having diabetes (Exact Odds Ratio [OR] = 11.79 (95% CI: 3.97, 37.26), p < 0.001). Analysis indicated area under curve (AUC) of ROC of 0.81 with 85.1% sensitivity and 67.3% specificity in distinguishing non-diabetic from diabetic subjects. The positive predictive value is 71.4%. CONCLUSION: lncRNA GAS5 levels are correlated to prevalence of T2DM. GENERAL SIGNIFICANCE: Assessment of GAS5 in serum along with other parameters offers greater accuracy in identifying individuals at-risk for diabetes.

Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells.

Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPARγ mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPARγ2 variant was spliced and when terminal differentiation occurs The appearance of PPARγ2 coordinates with the PPARγ1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPARγ1 and, primarily, PPARγ2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPARγ2, but not γ1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPARγ2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events.