PKO Mice Research Articles

SLC26A6 Deficiency Differentially Alters the Expression of MicroRNAs and RNA Binding Proteins in the Intestine

BACKGROUND: Putative anion transporter 1 (PAT1, SLC26A6), expressed at the luminal membrane of the intestinal epithelial cells, plays a key role in oxalate homeostasis and bicarbonate secretion. Additionally, we have recently shown that PAT1 knockout mice (PKO) exhibit compromised barrier integrity, gut dysbiosis and enhanced susceptibility to colitis. As defective PAT1 function and/or expression alters intestinal vulnerability to inflammation and miRNAs and RNA binding proteins are implicated in pathophysiology of gut inflammation, the focus of the current study was to investigate if loss of PAT1 modulates the expression of these post transcriptional regulators. Methods: microRNA was extracted from the colonic mucosa of age-matched PKO and wild type (WT) littermates (Qiagen miRNeasy mini kit) and subjected to miRNA expression profiling by Nanostring nCounter Mouse miRNA panel. Acquired raw data was further analyzed using the nSolver Analysis Software and ROSALIND online software platform to identify the differential gene expression. RT-PCR and western blotting was used to analyze the mRNA and protein expression of RNA binding proteins. Results: Genetic deficiency of PAT-1 resulted in differential expression of miRNAs compared to WT mice, where 60 miRNAs were notably upregulated, and 5 miRNAs were significantly downregulated in the colon. miR-150, known to be downregulated in ulcerative colitis patients, showed the maximum decrease (~6.9-fold change, P< 0.02) in PKO mice. Of note, miR-767 (known to promote tumor progression) was the top upregulated miRNA (~5.2-fold change, p<0.01). Interestingly, in silico analysis using TargetScan revealed that most of the upregulated miRNAs have been associated with the decrease in gut barrier function as previously shown in PKO mice. Further, a remarkable decrease in the protein expression of RNA binding proteins, HUR (0.82 ± 0.07 WT vs 0.49 + 0.03 PKO, P< 0.5) and CUGBP-1(0.37 ± 0.08 WT vs 0. 0.12 ± 0.03 PKO, P< 0.5) was also noted in PKO compared to WT mice. CONCLUSION: Our data provide evidence that PAT-1 deficiency in mice results in differential expression of several posttranscriptional regulators including miRNAs and RNA binding proteins with gene targets implicated in maintenance of epithelial barrier integrity. We speculate that altered expression of posttranscriptional regulators may potentially contribute to altered gut homeostasis observed in diseases associated with PAT1 deficiency. Supported by VA/NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acetyl-CoA carboxylase 1 deletion reduces platelet phospholipid and alters thrombus formation

Acetyl-CoA carboxylase (ACC) is the key enzyme regulating de novo lipogenesis (DNL). In recent years, ACC inhibitors has been of great interest to handle a wide range of diseases, especially cancers and cardiovascular diseases. However, the impact of its inhibition on hemostasis and thrombosis remains under-investigated. We previously demonstrated that ACC1 activation promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Here, we sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions and by decreasing phospholipid content. To test this hypothesis we generated a new Cre transgenic mouse strain in which ACC1 is specifically deleted in megakaryocyte and platelet (GpIbCre±x ACC1 flx/flx mouse). Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Platelet functions were assessed in vitro by aggregometry and ex vivo by flow chamber assay. Thrombus growth was analyzed in vivo with intravital microscopy. As expected, ACC1 deletion was restricted to megakaryocytic lineage. Hematological parameters show a platelet count decrease (30%) and a platelet volume increase (31%) in ACC1 knockout mice (ACC1 pKO) compared to the control mice. In agreement with our hypothesis, lipidomics analyses highlight a significant reduction in arachidonic acid containing phosphotidylethanolamine plasmalogen in ACC1 KO platelets. Logically, thromboxane A2 generation (upon thrombin or CRP stimulation) is also altered in ACC1 pKO mice in comparison to controls. This decrease in phospholipid content is associated with a default in thrombin and CRP-induced platelet aggregation and with impaired dense granules secretion. In line with these results, platelet-specific ACC1 deletion reduces thrombus growth and fibrinogen receptor activation. Platelet-specific ACC1 deletion leads to a decrease in phospholipid content which, in turn, decreases platelet thromboxane A2 generation, dense granules secretion and platelet aggregation. Thrombus formation is also reduced in platelet-specific ACC1 knockout mice, highlighting the importance of de novo lipogenesis and ACC-produced lipids in the regulation of platelet functions.

Inflammation and Calorie Restriction in Ercc1 PKO Mice

Inflammation and Calorie Restriction in Ercc1 PKO Mice

Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder

Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

Diabetic nephropathy in mice is aggravated by the absence of podocyte IRE1 and is correlated with reduced kidney ADH1 expression.

BackgroundInositol-requiring enzyme 1 (IRE1) plays a critical role in attenuating endoplasmic reticulum (ER) stress associated with renal injury which may also be a factor in diabetic nephropathy (DN). Alcohol dehydrogenase type I (ADH1) activity is prominent in the kidney, ADH1 activity is also reported to exert protective effects against ER stress that are not caused by alcohol consumption. However, the role of IRE1 in DN and the correlation between IRE1 and ADH1 activity remain unclear.MethodsIRE1α floxed mice (Ire1f/f) of C57BL/6J background were established and crossbred with Ire1αf/f mice to produce podocyte-specific IRE1α knockout mice. Male db/db mice (C57BLKS/J-leprdb/leprdb mice) were used as a DN model. Male mice were made diabetic by injection of streptozotocin. pLKO.1-based vectors encoding short hairpin RNA (shRNA) specific to the IRE1α gene were transfected into HEK293T cells to knockdown IRE1α in mouse podocytes. ELISA, Masson’s staining, and electron microscopy were performed to analyze the development of DN. The ADH1 expression was assayed by qPCR and western blot.ResultsWe found that IRE activity was increased in the glomeruli of DN mouse models. In contrast, ADH1 expression was decreased in these models and mice with podocyte-specific disruption of IRE1 (PKO mice). PKO mice that were made diabetic using strepto­zotocin exhibited accelerated proteinuria, enhanced glomerular fibrosis, and podocyte cell death. In addition, in cultured podocytes, the knockdown of IRE1 downregulated the ADH1 mRNA expression and induced ER stress, consistent with the result of PKO mice, while its detrimental effects were reversed by ADH1 overexpression.ConclusionsActivation of IRE1 in podocytes serves to limit the progress of DN. The dependence of kidney ADH1 expression on podocyte IRE1 further suggests that ADH1 activity may play an important role downstream of IRE1 in protecting against DN.

Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin-deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ Tcells and IFN-γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger-dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith . PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN-γ production by disease-inducing Tcells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger-dependence of FHL progression and revealed a higher contribution of CD4 Tcells and NK cells to IFN-γ production after MCMV infection. Importantly, therapeutic intervention by IFN-γ neutralization or CD8+ T-cell depletion had less benefit in MCMV-triggered FHL compared to LCMV-triggered FHL, likely due to MCMV-induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.

The Role of BTBD9 in the Cerebellum, Sleep-like Behaviors and the Restless Legs Syndrome

Recent genome-wide association studies (GWAS) have found cerebellum as a top hit for sleep regulation. Restless legs syndrome (RLS) is a sleep-related sensorimotor disorder characterized by uncomfortable sensations in the extremities, generally at night, which are often relieved by movements. Clinical studies have found that RLS patients have structural and functional abnormalities in the cerebellum. However, whether and how cerebellar pathology contributes to sleep regulation and RLS is not known. GWAS identified polymorphisms in BTBD9 conferring a higher risk of sleep disruption and RLS. Knockout of the BTBD9 homolog in mice (Btbd9) and fly results in motor restlessness and sleep disruption. We performed manganese-enhanced magnetic resonance imaging on the Btbd9 knockout mice and found decreased neural activities in the cerebellum, especially in lobules VIII, X, and the deep cerebellar nuclei. Electrophysiological recording of Purkinje cells (PCs) from Btbd9 knockout mice revealed an increased number of non-tonic PCs. Tonic PCs showed increased spontaneous activity and intrinsic excitability. To further investigate the cerebellar contribution to RLS and sleep-like behaviors, we generated PC-specific Btbd9 knockout mice (Btbd9 pKO) and performed behavioral studies. Btbd9 pKO mice showed significant motor restlessness during the rest phase but not in the active phase. Btbd9 pKO mice also had an increased probability of waking at rest. Unlike the Btbd9 knockout mice, there was no increased thermal sensation in the Btbd9 pKO. Our results indicate that the Btbd9 knockout influences the PC activity; dysfunction in the cerebellum may contribute to the motor restlessness found in the Btbd9 knockout mice.

NF-kB1 induces T cell dysfunction to promote colon tumorigenesis

Abstract Background and aims Chronic colonic inflammation drives colon tumorigenesis. Chronic infection-associated inflammation causes T cell exhaustion/dysfunction. NF-kB is a molecular link between colonic inflammation and colorectal cancer. We aimed at testing the hypothesis that the p50 NF-kB (NF-kB1) regulates T cell dysfunction to promote colon tumor immune evasion and development. Methods Nfkb1 knock out (p50 KO), Nfkb1 KO chimera mice, Nfkb1 KO colon tumor cells were used to determine p50 NF-kB function in colon tumor development. RNA-Seq, chromatin immunoprecipitation, and electrophoresis mobility shift assay were used to elucidate molecular mechanism of NF-kB1 action. Flow cytometry was used to analyze cytotoxic T lymphocytes. Results p50 KO mice showed a significantly decrease in tumorigenesis. However, mice with p50 deficiency only in immune cells also exhibited a significant decrease in tumorigenesis, and p50-sufficient colon tumor cells also grew significantly slower in p50 KO mice than in WT mice. RNA-Seq identified Gzmb as a p50 NF-kB target gene. p50 KO mice have significantly higher tumor-infiltrating granzyme B+ CTLs than WT mice, and CTL granzyme B protein level is also significantly higher in p50 KO mice than in WT mice. A kB sequence element was identified at the Gzmb promoter and p50 NF-kB directly binds to this kB element in T cells to repress Gzmb expression. Conclusion NF-kB1 has opposing functions in tumor cells and T cells in vivo. The p50 NF-kB represses Gzmb expression to confer colon tumor-infiltrating CTL a dysfunctional phenotype to promote colon tumor immune evasion, resulting in enhanced colon tumorigenesis and growth.

Sirt6 in pro-opiomelanocortin neurons controls energy metabolism by modulating leptin signaling

ObjectiveSirt6 is an essential regulator of energy metabolism in multiple peripheral tissues. However, the direct role of Sirt6 in the hypothalamus, specifically pro-opiomelanocortin (POMC) neurons, controlling energy balance has not been established. Here, we aimed to determine the role of Sirt6 in hypothalamic POMC neurons in the regulation of energy balance and the underlying mechanisms. MethodsFor overexpression studies, the hypothalamic arcuate nucleus (ARC) of diet-induced obese mice was targeted bilaterally and adenovirus was delivered by using stereotaxic apparatus. For knockout studies, the POMC neuron-specific Sirt6 knockout mice (PKO mice) were generated. Mice were fed with chow diet or high-fat diet, and body weight and food intake were monitored. Whole-body energy expenditure was determined by metabolic cages. Parameters of body composition and glucose/lipid metabolism were evaluated. ResultsSirt6 overexpression in the ARC ameliorated diet-induced obesity. Conversely, selective Sirt6 ablation in POMC neurons predisposed mice to obesity and metabolic disturbances. PKO mice showed an increased fat mass and food intake, while the energy expenditure was decreased. Mechanistically, Sirt6 could modulate leptin signaling in hypothalamic POMC neurons, with Sirt6 deficiency impairing leptin-induced phosphorylation of signal transducer and activator of transcription 3. The effects of leptin on reducing food intake and body weight and leptin-stimulated lipolysis were also impaired. Moreover, Sirt6 inhibition diminished the leptin-induced depolarization of POMC neurons. ConclusionsOur results reveal a key role of Sirt6 in POMC neurons against energy imbalance, suggesting that Sirt6 is an important molecular regulator for POMC neurons to promote negative energy balance.

Loss of mural cell-derived laminin aggravates hemorrhagic brain injury

BackgroundMural cells synthesize and deposit laminin to the basement membrane. To investigate the function of mural cell-derived laminin, we generated a mutant mouse line lacking mural cell-derived laminin (termed PKO). In a previous study, we showed that the PKO mice were grossly normal under homeostatic condition, but developed blood-brain barrier (BBB) breakdown with advanced age (> 8 months), suggesting that these mutants are intrinsically weak. Based on these findings, we hypothesized that PKO mice have exacerbated injuries in pathological conditions.MethodsUsing collagenase-induced intracerebral hemorrhage (ICH) as an injury model, we examined various stroke outcomes, including hematoma volume, neurological function, neuronal death, BBB integrity, paracellular/transcellular transport, inflammatory cell infiltration, and brain water content, in PKO mice and their wildtype littermates at young age (6–8 weeks). In addition, transmission electron microscopy (TEM) analysis and an in vitro ICH model were used to investigate the underlying molecular mechanisms.ResultsCompared to age-matched wildtype littermates, PKO mice display aggravated stroke outcomes, including larger hematoma size, worse neurological function, increased neuronal cell death, enhanced BBB permeability, increased transcytosis, and elevated inflammatory cell infiltration. These mutants also exhibit high baseline brain water content independent of aquaporin-4 (AQP4). In addition, mural cell-derived laminin significantly reduced caveolin-1 without affecting tight junction proteins in the in vitro ICH model.ConclusionsThese results suggest that mural cell-derived laminin attenuates BBB damage in ICH via decreasing caveolin-1 and thus transcytosis, regulates brain water homeostasis, and plays a beneficial role in ICH.

Ultrasonic communication deficits in p50 KO mice and effect of maternal enrichment

Ultrasonic communication deficits in p50 KO mice and effect of maternal enrichment

Specific profile of ultrasonic communication in a mouse model of neurodevelopmental disorders

Mice emit ultrasonic vocalizations (USVs) in different social conditions: pups maternal separation, juveniles play, adults mating and social investigation. The USVs measurement has become an important instrument for behavioural phenotyping in neurodevelopmental disorders (NDDs). Recently, we have demonstrated that the deletion of the NFκB1 gene, which encodes the p50 NF-κB subunit, causes NDDs phenotype in mice. In this study, we investigated the ultrasonic communication and the effects of an early social enrichment in mice lacking the NF-κB p50 subunit (p50 KO). In particular, USVs of wild-type (WT), p50 KO and KO exposed to early social enrichment (KO enriched) were recorded using an ultrasound sensitive microphone and analysed by Avisoft software. USVs analysis showed that p50 KO pups emit more and longer vocalizations compared to WT pups. On the contrary, in adulthood, p50 KO mice emit less USVs than WT mice. We also found significant qualitative differences in p50 KO mice USVs compared to WT mice; the changes specifically involved two USVs categories. Early social enrichment had no effect on USVs number, duration and type in p50 KO mice. Together, these data revealed social communication alterations in a mouse model of NDDs; these deficits were not recovered by early social enrichment, strengthening the fact that genetic background prevails on environmental enrichment.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3.

BackgroundProtein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated.MethodsPancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells.ResultsPrmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3.ConclusionPRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Abstract TP341: Pericyte Derived Laminin Play a Beneficial Role in Intracerebral Hemorrhage

Introduction: Laminin, a major component of the basement membrane, exists in many isoforms. Although loss of laminin is found in intracerebral hemorrhage (ICH), the exact biological function of laminin in ICH remains largely unknown. This is primarily due to its intrinsic complexity (various isoforms) and the lack of genetic tools. In a previous study, we generated a transgenic line with laminin deficiency specifically in pericytes (PKO). These PKO mice are grossly normal in pure C57Bl6 background at young age, although mild blood brain barrier (BBB) breakdown is detected in aged mutants. In this study, we aim to investigate the role of pericyte-derived laminin in brain pathology and disease progression in ICH. Methods: Collagenase was injected into the striatum of young (2-month-old) control and PKO mice to induce hemorrhage. At 2, 5, and 14 days post injury (dpi), hematoma size, neuronal death, vascular permeability, inflammatory cell infiltration, and neurological deficits were examined. Results: Compared to the controls, the PKO mice showed larger hematoma and increased neuronal deaths at 2 and 5 dpi, but more severe neurological deficits at 14 dpi. Additionally, substantially increased IgG level was found in PKO brains at 14 dpi, indicating BBB integrity disruption. Consistent with this observation, significantly higher levels of CD45 + leukocytes and Ly6G + granulocytes were detected in PKO brains at 14 dpi. Similarly, elevated CD3 staining was found in PKO brains at 5 and 14 dpi. These data suggest that the recovery of vascular integrity after ICH is delayed in PKO mice. Moreover, PKO brains also showed increased number of CD11b + microglia/macrophages at all three time points examined, suggesting a possible role of pericyte-derived laminin in microglia/macrophage activation and/or migration. Conclusion: Our data support a neuroprotective role of pericyte-derived laminin in ICH. This beneficial effect is probably due to its biological activities in: (1) promoting neuronal survival; (2) stabilizing newly formed vessels and enhancing their integrity; (3) inhibiting inflammatory cell infiltration; and (4) regulating the activation and/or migration of microglia/macrophages.

Abstract 218: Pericytic Laminin Regulates Blood-Brain Barrier Integrity in an Age-Dependent Manner

Introduction: Laminin, a major component of the basement membrane, plays an important role in blood brain barrier (BBB) regulation. At the neurovascular unit, astrocytes, brain endothelial cells, and pericytes synthesize and deposit different laminin isoforms into the basement membrane. Previous studies from our laboratory showed that loss of astrocytic laminin induces age-dependent and region-specific BBB breakdown and intracerebral hemorrhage, suggesting a critical role of astrocytic laminin in vascular integrity maintenance. Laminin α4 (predominantly generated by endothelial cells) has been shown to regulate vascular integrity at embryonic/neonatal stage. The role of pericytic laminin in vascular integrity, however, remains elusive. Methods: We investigated the function of pericyte-derived laminin in vascular integrity using laminin conditional knockout mice. Specifically, laminin floxed mice were crossed with PDGFRβ-Cre line to generate mutants (PKO) with laminin deficiency in PDGFRβ + cells, which include both pericytes and vascular smooth muscle cells (vSMCs). To distinguish the contribution of pericyte- and vSMC-derived laminin, we also generated a vSMC-specific condition knockout line (TKO) by crossing the laminin floxed mice with Transgelin-Cre mice. In this study, mice of both genders on a C57Bl6 background were used. At least 5-6 animals were used in biochemical and histological analyses in this study. Results: Pericyte-derived laminin was abrogated in all PKO mice. However, only old but not young PKO mice showed signs of BBB breakdown and reduced vessel density, suggesting age-dependent changes. Consistent with these data, further mechanistic studies revealed reduced tight junction proteins, diminished AQP4 expression, and deceased pericyte coverage in old but not young PKO mice. In addition, neither BBB disruption nor decreased vessel density was observed in TKO mice, suggesting that these vascular defects are due to loss of pericyte- rather than vSMC-derived laminin. Conclusions: These results strongly suggest that pericyte-derived laminin active regulates BBB integrity and vessel density in an age-dependent manner. I would like this abstract to be considered for the Stroke Basic Science Award.

Properdin deficiency protects from 5-fluorouracil-induced small intestinal mucositis in a complement activation-independent, interleukin-10-dependent mechanism.

Intestinal mucositis is a serious complication of chemotherapy that leads to significant morbidity that may require dose or drug adjustments. Specific mitigating strategies for mucositis are unavailable, due partly to an incomplete understanding of the pathogenic mechanisms. We have previously shown an effect of properdin, a positive regulator of complement activation, in models of colitis. Here we use properdin-deficient (PKO ) mice to interrogate the role of properdin and complement in small intestinal mucositis. Mucositis was induced by five daily injections of 5-fluorouracil (5-FU) in wild-type (WT), PKO , interleukin (IL)-10-/- and properdin/IL-10-/- double knock-out (DKO) mice. At the time of euthanasia their jejunum was collected for histology, immunohistochemistry and cytokine and complement activation measurements. Complement became activated in mice receiving 5-FU, indicated by increased intestinal levels of C3a and C5a. Compared to WT, PKO mice experienced significantly less mucositis, despite C3a levels as high as inflamed WT mice and slightly less C5a. Conversely, PKO mice had higher intestinal levels of IL-10. IL-10 expression was mainly by epithelial cells in both uninflamed and inflamed PKO mice. IL-10-/- mice proved to be highly susceptible to mucositis and DKO mice were equally susceptible, demonstrating that a lack of properdin does not protect mice lacking IL-10. We interpret our findings to indicate that, to a significant extent, the inflammation of mucositis is properdin-dependent but complement activation-independent. Additionally, the benefit achieved in the absence of properdin is associated with increased IL-10 levels, and IL-10 is important in limiting mucositis.

Novel insights into the role of NF-κB p50 in astrocyte-mediated fate specification of adult neural progenitor cells.

Within the CNS nuclear factor-kappa B (NF-κB) transcription factors are involved in a wide range of functions both in homeostasis and in pathology. Over the years, our and other groups produced a vast array of information on the complex involvement of NF-κB proteins in different aspects of postnatal neurogenesis. In particular, several extracellular signals and membrane receptors have been identified as being able to affect neural progenitor cells (NPC) and their progeny via NF-κB activation. A crucial role in the regulation of neuronal fate specification in adult hippocampal NPC is played by the NF-κB p50 subunit. NF-κB p50KO mice display a remarkable reduction in adult hippocampal neurogenesis which correlates with a selective defect in hippocampal-dependent short-term memory. Moreover absence of NF-κB p50 can profoundly affect the in vitro proneurogenic response of adult hippocampal NPC (ahNPC) to several endogenous signals and drugs. Herein we briefly review the current knowledge on the pivotal role of NF-κB p50 in the regulation of adult hippocampal neurogenesis. In addition we discuss more recent data that further extend the relevance of NF-κB p50 to novel astroglia-derived signals which can influence neuronal specification of ahNPC and to astrocyte-NPC cross-talk.

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF-κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF-κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell-autonomous and noncell-autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC-MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin-2 (LCN-2) was recognized as a novel astroglial-derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN-2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN-2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin-1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC-astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169-181.

Loss of pancreatic Mig6 improves glucose homeostasis during diet‐induced obesity

In response to nutritional overload, functional beta cell mass normally expands to compensate for diet‐induced insulin resistance, thereby preserving glucose homeostasis. In the pathogenesis of type 2 diabetes (T2D), however, the compensation is insufficient, as beta cell dysfunction and destruction develop, leading to a mismatch between functional beta cell mass and insulin sensitivity. Thus, targeting beta cell proliferation as well as beta cell secretory capacity (i.e., function) and survival could prevent the onset of the disease. We have been investigating the impact of the elevated levels of mitogen inducible gene 6 (Mig6) in beta cells during glucolipotoxicity, as this milieu is thought to contribute to T2D. Mig6 is an adaptor protein that impairs EGFR signaling, a critical cell survival and proliferation pathway. Thus, we hypothesized that ablating Mig6 would fortify functional beta cell mass and preserve glucose homeostasis during diet‐induced obesity and insulin resistance. To this end, we compared mice lacking pancreatic Mig6 (PKO) and their wild‐type (WT) littermates fed either a low fat or high fat diet. No observable phenotypic differences were observed between either genotype fed the low fat diet. High fat feeding increased body mass and decreased glucose tolerance in WT mice, despite a robust increase in beta cell cross‐sectional area (as an index of beta cell mass). In contrast, high fat‐fed PKO mice had improved glucose tolerance compared to WT littermates, despite no increase in beta cell cross‐sectional area. These data suggest that either PKO mice are protected from diet‐induced insulin resistance or their beta cell function is enhanced to the extent to be able to compensate for systemic insulin resistance. Therefore, blocking the actions of Mig6 and thereby enhancing EGFR signaling could represent a novel strategy for preventing or delaying the progression to T2D.Support or Funding InformationNIH grant DK099311 (PTF)