Circulating Exosomes from Alzheimer's Disease Suppress Vascular Endothelial-Cadherin Expression and Induce Barrier Dysfunction in Recipient Brain Microvascular Endothelial Cell.

BACKGROUND: Blood-brain barrier (BBB) breakdown is a crucial aspect of Alzheimer's disease (AD) progression. Dysfunction in BBB is primarily caused by impaired tight junction and adherens junction proteins in brain microvascular endothelial cells (BMECs). The role of adherens junctions in AD-related BBB dysfunction remains unclear. Exosomes from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells. However, it remains unknown if and how these exosomes cause BMEC dysfunction in AD. OBJECTIVE: This study aimed to investigate the impact of AD circulating exosomes on brain endothelial dysfunction. METHODS: Exosomes were isolated from sera of AD patients and age- and sex-matched cognitively normal controls using size-exclusion chromatography. The study measured the biomechanical nature of BMECs' endothelial barrier, the lateral binding forces between live BMECs. Paracellular expressions of the key adherens junction protein vascular endothelial (VE)-cadherin were visualized in BMEC cultures and a 3D BBB model using human BMECs and pericytes. VE-cadherin signals were also examined in brain tissues from AD patients and normal controls. RESULTS: Circulating exosomes from AD patients reduced VE-cadherin expression levels and impaired barrier function in recipient BMECs. Immunostaining analysis demonstrated that AD exosomes damaged VE-cadherin integrity in a 3D microvascular tubule formation model. The study found that AD exosomes weakened BBB integrity depending on their RNA content. Additionally, diminished microvascular VE-cadherin expression was observed in AD brains compared to controls. CONCLUSION: These findings highlight the significant role of circulating exosomes from AD patients in damaging adherens junctions of recipient BMECs, dependent on exosomal RNA.

Circulating exosomes from Alzheimer's disease suppress VE-cadherin expression and induce barrier dysfunction in recipient brain microvascular endothelial cell.

Background: Blood-brain barrier (BBB) breakdown is a component of the progression and pathology of Alzheimer's disease (AD). BBB dysfunction is primarily caused by reduced or disorganized tight junction or adherens junction proteins of brain microvascular endothelial cell (BMEC). While there is growing evidence of tight junction disruption in BMECs in AD, the functional role of adherens junctions during BBB dysfunction in AD remains unknown. Exosomes secreted from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells. However, it remains unknown if and how these exosomes cause BMEC dysfunction in AD. Objectives: This study aimed to investigate the potential roles of AD circulating exosomes and their RNA cargos in brain endothelial dysfunction in AD. Methods: We isolated exosomes from sera of five cases of AD compared with age- and sex-matched cognitively normal controls using size-exclusion chromatography technology. We validated the qualities and particle sizes of isolated exosomes with nanoparticle tracking analysis and atomic force microscopy. We measured the biomechanical natures of the endothelial barrier of BMECs, the lateral binding forces between live BMECs, using fluidic force miscopy. We visualized the paracellular expressions of the key adherens junction protein VE-cadherin in BMEC cultures and a 3D BBB model that employs primary human BMECs and pericytes with immunostaining and evaluated them using confocal microscopy. We also examined the VE-cadherin signal in brain tissues from five cases of AD and five age- and sex-matched cognitively normal controls. Results: We found that circulating exosomes from AD patients suppress the paracellular expression levels of VE-cadherin and impair the barrier function of recipient BMECs. Immunostaining analysis showed that AD circulating exosomes damage VE-cadherin integrity in a 3D model of microvascular tubule formation. We found that circulating exosomes in AD weaken the BBB depending on the RNA cargos. In parallel, we observed that microvascular VE-cadherin expression is diminished in AD brains compared to normal controls. Conclusion: Using in vitro and ex vivo models, our study illustrates that circulating exosomes from AD patients play a significant role in mediating the damage effect on adherens junction of recipient BMEC of the BBB in an exosomal RNA-dependent manner. This suggests a novel mechanism of peripheral senescent exosomes for AD risk.

CD31 as a probable responding and gate-keeping protein of the blood-brain barrier and the risk of Alzheimer's disease.

Several studies have shown that an abnormal vascular-immunity link could increase Alzheimer's disease (AD) risk; however, the mechanism is unclear. CD31, also named platelet endothelial cell adhesion molecule (PECAM), is a surface membrane protein of both endothelial and immune cells and plays important roles in the interaction between the vascular and immune systems. In this review, we focus on research regarding CD31 biological actions in the pathological process that may contribute to AD based on the following rationales. First, endothelial, leukocyte and soluble forms of CD31 play multi-roles in regulating transendothelial migration, increasing blood-brain barrier (BBB) permeability and resulting in neuroinflammation. Second, CD31 expressed by endothelial and immune cells dynamically modulates numbers of signaling pathways, including Src family kinases, selected G proteins, and ß-catenin which in turn affect cell-matrix and cell-cell attachment, activation, permeability, survival, and ultimately neuronal cell injury. In endothelia and immune cells, these diverse CD31-mediated pathways act as a critical regulator in the immunity-endothelia-brain axis, thereby mediating AD pathogenesis in ApoE4 carriers, which is the major genetic risk factor for AD. This evidence suggests a novel mechanism and potential drug target for CD31 in the background of genetic vulnerabilities and peripheral inflammation for AD development and progression.

Peripheral apolipoprotein E proteins and their binding to LRP1 antagonize Alzheimer's disease pathogenesis in the brain during peripheral chronic inflammation.

C-reactive protein (CRP) impacts apolipoprotein E4 (ApoE4) allele to increase Alzheimer's disease (AD) risk. However, it is unclear how the ApoE protein and its binding to LRP1 are involved. We found that ApoE2 carriers had the highest but ApoE4 carriers had the lowest concentrations of blood ApoE in both humans and mice; blood ApoE concentration was negatively associated with AD risk. Elevation of peripheral monomeric CRP (mCRP) reduced the expression of ApoE in ApoE2 mice, while it decreased ApoE-LRP1 binding in the brains of ApoE4 mice that was characterized by Proximity Ligation Assay. Both serum ApoE and brain ApoE-LRP1 binding were positively associated with the expression of pericytes that disappeared after mCRP treatment, and negatively associated with brain tauopathy and neuroinflammation in the presence of mCRP. In ApoE-/- mice, mCRP reduced the brain expression levels of synaptophysin and PSD95 and the positive relationship between ApoE-LRP1 binding and synaptophysin or PSD95 expression disappeared. Our study suggests that blood ApoE protects against AD pathogenesis by binding to LRP1 during peripheral chronic inflammation.

Monomeric C-reactive protein induces the cellular pathology of Alzheimer's disease.

Introduction: Human study shows that elevated C-reactive protein (CRP) in blood impacts apolipoprotein E (APOE) ε4, but not APOE ε3 or APOE ε2, genotype to increase the risk of Alzheimer's disease (AD). However, whether CRP is directly involved in cellular AD pathogenesis and in which type of neuronal cells of APOE ε4 carriers are unknown. Methods: We aimed to use different primary neuronal cells and investigate if CRP induces cellular AD pathology depending on APOE genotypes. Here the different primary neuronal cells from the different APOE genotype knock-in mice cortex were isolated and used. Results: Monomeric CRP (mCRP) increased amyloid beta production and, in parallel, induced tau phosphorylation in addition to their related proteins in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose- and time-dependent manner. Consistently, mCRP induced the staining of other neurodegenerative biomarkers, including Fluoro-Jade B stain (FjB), TUNEL and Cleaved Caspase-3, in primary neurons in a similar pattern of APOE ε4 > APOE ε3 > APOE ε2. In contrast, pentameric CRP (pCRP) had a tendency to induce cellular AD pathology but did not reach statistical significance. On the other hand, it is intriguing that regardless of APOE genotype, mCRP did not influence the expressions of Iba-1 and CD68 in primary microglia or the expression of glial fibrillary acidic protein in primary astrocytes, and additionally mCRP did not affect the secretions of interleukin (IL)-1α, IL-1ß, and tumor necrosis factor α from these cells. Discussion: This is the first report to demonstrate that mCRP directly induces cellular AD pathogenesis in neurons in an APOE genotype-dependent pattern, suggesting that mCRP plays a role as a mediator involved in the APOE ε4-related pathway for AD during chronic inflammation. Highlights: Pentameric C-reactive protein (pCRP) can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP) during and after the acute phase.mCRP increased amyloid beta production in the primary neurons in a pattern of apolipoprotein E (APOE) ε4 > APOE ε3 > APOE ε2 in a dose-dependent manner.mCRP induced the expression of phosphorylated tau in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose- and time-dependent manner.mCRP plays an important mediator role in the APOE ε4-related pathway of Alzheimer's disease risk.

Monomeric C-reactive protein via endothelial CD31 for neurovascular inflammation in an ApoE genotype-dependent pattern: A risk factor for Alzheimer's disease?

In chronic peripheral inflammation, endothelia in brain capillary beds could play a role for the apolipoprotein E4 (ApoE4)-mediated risk for Alzheimer's disease (AD) risk. Using human brain tissues, here we demonstrate that the interactions of endothelial CD31 with monomeric C-reactive protein (mCRP) versus ApoE were linked with shortened neurovasculature for AD pathology and cognition. Using ApoE knock-in mice, we discovered that intraperitoneal injection of mCRP, via binding to CD31 on endothelial surface and increased CD31 phosphorylation (pCD31), leading to cerebrovascular damage and the extravasation of T lymphocytes into the ApoE4 brain. While mCRP was bound to endothelial CD31 in a dose- and time-dependent manner, knockdown of CD31 significantly decreased mCRP binding and altered the expressions of vascular-inflammatory factors including vWF, NF-κB and p-eNOS. RNAseq revealed endothelial pathways related to oxidative phosphorylation and AD pathogenesis were enhanced, but endothelial pathways involving in epigenetics and vasculogenesis were inhibited in ApoE4. This is the first report providing some evidence on the ApoE4-mCRP-CD31 pathway for the cross talk between peripheral inflammation and cerebrovasculature leading to AD risk.

Oral Amylin Treatment Reduces the Pathological Cascade of Alzheimer's Disease in a Mouse Model.

Intraperitoneal injection of amylin or its analog reduces Alzheimer's disease (AD) pathology in the brains. However, self-injecting amylin analogs is difficult for patients due to cognitive deficits. This work aims to study the effects of amylin on the brain could be achieved by oral delivery as some study reported that amylin receptor may be present in the gastrointestinal tract. A 6-week course of oral amylin treatment reduced components of AD pathology, including the levels of amyloid-ß, phosphorylated tau, and ionized calcium binding adaptor molecule 1. The treatment reduced active forms of cyclin-dependent kinase 5. Oral amylin treatment led to improvements in social deficit in AD mouse. Using immunofluorescence, we observed the amylin receptor complexed with the calcitonin receptor and receptor activity-modifying proteins in the enteric neurons. The study suggests the potential of the oral delivery of amylin analogs for the treatment of AD and other neurodegenerative diseases through enteric neurons.

A randomized controlled trial of exercise on augmenting the effects of cognitive remediation in persons with severe mental illness.

BACKGROUND: Preliminary evidence suggests that aerobic exercise may augment the effects of cognitive remediation on improving cognitive functioning in severe mental illness. It has also been hypothesized that increases in cognitive functioning associated with adding exercise are mediated by increases in brain derived neurotrophic factor (BDNF). However, rigorous controlled trials are lacking. METHODS: A randomized controlled trial was conducted to explore whether adding a 30-h aerobic exercise program over 10 weeks to an equally intensive cognitive remediation program (CR + E) improved cognitive functioning more than cognitive remediation alone (CR-Only). Thirty-four participants with schizophrenia or bipolar disorder were randomly assigned to CR + E or CR-Only, and cognitive functioning was assessed at baseline and post-treatment. Total and mature BDNF were measured in blood serum at baseline, Week-5 pre- and post-exercise, and Week-10 pre- and post-exercise. RESULTS: Participants in both conditions had high levels of engagement in the interventions and improved significantly in cognitive functioning, but did not differ in amount of cognitive change. The groups also did not differ in changes in BDNF from pre-to post-exercise at Weeks 5 or 10, nor in resting BDNF levels. Exploratory analyses indicated that higher body mass index (BMI) significantly predicted attenuated improvement in cognitive functioning for both groups. DISCUSSION: Exercise did not augment the effects of cognitive remediation in persons with severe mental illness, possibly because the cognitive remediation program resulted in strong gains in cognitive functioning. Moderate aerobic exercise does not appear to reliably increase BDNF levels in persons with severe mental illness. CLINICALTRIALS. GOV IDENTIFIER: NCT02326389.

Characterization of the effects of calcitonin gene-related peptide receptor antagonist for Alzheimer's disease.

Calcitonin gene-related peptide (cGRP) receptor antagonists effectively treat migraine through reducing neuroinflammation, vasoconstriction and possibly neruogenesis. Since neuroinflammation is also involved in the pathogenesis of Alzheimer's diseases (AD), we hypothesized and tested if a cGRP receptor antagonist, BIBN 4096 BS (BIBN), has effects on AD pathology. Using an AD mouse model, 5XFAD, with different ages, here we report that the BIBN treatment significantly increased the brain expression of PSD95, a postsynaptic protein, in both young and old AD mice. In parallel, BIBN improved learning and memory in the behavior test in the young, but not old, AD mice. The BIBN treatment reduced α-synuclein aggregation in both young and old AD mice. BIBN significantly decreased neuroinflammatory markers of ionized calcium binding adapter molecules-1 (Iba-1) and the p38 MAPK and NFκB signaling pathways in young, but not old, AD mice. The treatment also reduced the accumulation of amyloid-ß (Aß), and decreased tau phosphorylation through the pathway of CDK5/p25 in young mice only. Our study provides the evidence and suggests that the cGRP antagonists might be a therapeutic target to attenuate the pathological cascade and delay cognitive decline of AD in humans.

Olig2 regulates terminal differentiation and maturation of peripheral olfactory sensory neurons.

The bHLH transcription factor Olig2 is required for sequential cell fate determination of both motor neurons and oligodendrocytes and for progenitor proliferation in the central nervous system. However, the role of Olig2 in peripheral sensory neurogenesis remains unknown. We report that Olig2 is transiently expressed in the newly differentiated olfactory sensory neurons (OSNs) and is down-regulated in the mature OSNs in mice from early gestation to adulthood. Genetic fate mapping demonstrates that Olig2-expressing cells solely give rise to OSNs in the peripheral olfactory system. Olig2 depletion does not affect the proliferation of peripheral olfactory progenitors and the fate determination of OSNs, sustentacular cells, and the olfactory ensheathing cells. However, the terminal differentiation and maturation of OSNs are compromised in either Olig2 single or Olig1/Olig2 double knockout mice, associated with significantly diminished expression of multiple OSN maturation and odorant signaling genes, including Omp, Gnal, Adcy3, and Olfr15. We further demonstrate that Olig2 binds to the E-box in the Omp promoter region to regulate its expression. Taken together, our results reveal a distinctly novel function of Olig2 in the periphery nervous system to regulate the terminal differentiation and maturation of olfactory sensory neurons.

Association of Plasma Amylin Concentration With Alzheimer Disease and Brain Structure in Older Adults.

Importance: Preclinical studies suggest that amylin has a U-shaped dose-response association with risk of Alzheimer disease (AD). The association of plasma amylin with AD in humans is unknown. Objectives: To measure amylin concentration in plasma by using enzyme-linked immunosorbent assay and to study the association between plasma amylin, incidence of AD, and brain structure in humans. Design, Setting, and Participants: This cohort study used data from the Framingham Heart Study offspring cohort from 1998 to 2015. Using a Monte Carlo approach, participants were divided into 3 plasma amylin concentration groups: (1) low (<75 pmol/L), (2) high (75-2800 pmol/L), and (3) extremely high (≥2800 pmol/L). Data analyses were conducted October 5, 2017, to December 18, 2018. Exposures: Baseline plasma amylin concentrations at examination 7. Main Outcomes and Measures: Incidence of dementia or AD and brain volumetric measures from structural magnetic resonance imaging data. Results: From the Framingham Heart Study offspring cohort, 3061 participants (mean [SD] age at baseline, 61.0 [9.5] years; 1653 [54.0%] women) who had plasma amylin measurements, dementia incidence, and brain volume measurements on record were included in this study. The distribution of plasma amylin concentrations was highly skewed (median [interquartile range], 7.5 [4.6-18.9] pmol/L; mean [SD], 302.3 [1941.0] pmol/L; range, 0.03-44 623.7 pmol/L). Compared with the low plasma amylin concentration group, the high plasma amylin concentration group had a lower rate of AD incidence (2.3% vs 5.6%; P = .04), but the extremely high plasma amylin concentration group had a higher rate of AD incidence (14.3%; P < .001). After adjusting for age, sex, education, body mass index, diabetes, cardiovascular disease, high-density lipoprotein level, and APOE4, high plasma amylin was not associated with decreased AD risk (hazard ratio, 0.42 [95% CI, 0.16-1.14]; P = .09) but was positively associated with volume of gray matter in the temporal lobe (ß = 0.17 [SE, 0.05]; P < .001). In contrast, extremely high plasma amylin concentration was associated with a higher AD risk (hazard ratio, 2.51 [95% CI, 1.38-4.57]; P = .003) but not associated with temporal lobe volume (ß = 0.02 [SE, 0.07]; P = .82). Conclusions and Relevance: This study found that plasma amylin concentration was associated with AD incidence and brain structure with a U-shaped pattern. These findings are consistent with preclinical findings that suggest amylin is a neuropeptide that is physiological; however, at extremely high concentrations, it may lead to amylin aggregation and therefore may be a risk factor for AD.

Effects of Amylin Against Amyloid-ß-Induced Tauopathy and Synapse Loss in Primary Neurons.

Recent studies demonstrate that peripheral amylin treatment reduces pathology in mouse models of Alzheimer's disease (AD). However, soluble and aggregated amylin are distinct species; while amylin is a physiological neuropeptide, amylin aggregation is a pathological factor for diabetes. We thus hypothesized that because of their similarity in secondary structures, amylin antagonizes amyloid-ß peptide (Aß)-induced AD pathology in neurons with a dose-dependent pattern. To test the hypothesis, we conducted both in vitro and in vivo experiments with different doses of amylin and with its analog, pramlintide. Here we report that a high concentration of either Aß or amylin alone induced tau phosphorylation (pTau) in primary neurons. Interestingly, with a low concentration, amylin had direct effects to reverse the Aß-induced pTau, as well as damaged neuronal synapses and neurite disorganization. However, when the concentration was high (10.24 µM), amylin lost the effects against the Aß-induced cellular AD pathology and, together with Aß, worsened tauopathy in neurons. In the 5XFAD AD mouse model, daily peripheral amylin treatment with a low dose (200 µg/kg) more effectively reduced amyloid burden, and increased synapse, but with a high dose (800 µg/kg), it more effectively reduced tauopathy. Correspondingly, amylin treatment improved learning and memory in these mice. It demonstrates that amylin has a dose-dependent U-shape effect against AD pathogenesis. Within a physiological range, amylin is a neuroprotective hormone against AD in neurons; but when both Aß and amylin concentrations are elevated, imbalance of Aß and amylin may contribute to brain AD pathogenesis.

Pax6 mediates ß-catenin signaling for self-renewal and neurogenesis by neocortical radial glial stem cells.

The Wnt/ß-catenin pathway is a critical stem cell regulator and plays important roles in neuroepithelial cells during early gestation. However, the role of Wnt/ß-catenin signaling in radial glia, a major neural stem cell population expanded by midgestation, remains poorly understood. This study shows that genetic ablation of ß-catenin with hGFAP-Cre mice inhibits neocortical formation by disrupting radial glial development. Reduced radial glia and intermediate progenitors are found in the ß-catenin-deficient neocortex during late gestation. Increased apoptosis and divergent localization of radial glia in the subventricular zone are also observed in the mutant neocortex. In vivo and in vitro proliferation and neurogenesis as well as oligodendrogenesis by cortical radial glia or by dissociated neural stem cells are significantly defective in the mutants. Neocortical layer patterning is not apparently altered, while astrogliogenesis is ectopically increased in the mutants. At the molecular level, the expression of the transcription factor Pax6 is dramatically diminished in the cortical radial glia and the sphere-forming neural stem cells of ß-catenin-deficient mutants. Chromatin immunoprecipitation and luciferase assays demonstrate that ß-catenin/Tcf complex binds to Pax6 promoter and induces its transcriptional activities. The forced expression of Pax6 through lentiviral transduction partially rescues the defective proliferation and neurogenesis by ß-catenin-deficient neural stem cells. Thus, Pax6 is a novel downstream target of the Wnt/ß-catenin pathway, and ß-catenin/Pax6 signaling plays critical roles in self-renewal and neurogenesis of radial glia/neural stem cells during neocortical development.

ß-catenin regulates Pax3 and Cdx2 for caudal neural tube closure and elongation.

Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/ß-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of ß-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/ß-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/ß-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the ß-catenin mutants, suggesting that Pax3 is a key downstream effector of ß-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the ß-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both ß-catenin and Pax3 is required for regional Cdx2 expression. Thus, ß-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP.

Canonical Wnt signaling promotes the proliferation and neurogenesis of peripheral olfactory stem cells during postnatal development and adult regeneration.

The mammalian olfactory epithelium (OE) has a unique stem cell or progenitor niche, which is responsible for the constant peripheral neurogenesis throughout the lifespan of the animal. However, neither the signals that regulate the behavior of these cells nor the lineage properties of the OE stem cells are well understood. Multiple Wnt signaling components exhibit dynamic expression patterns in the developing OE. We generated Wnt signaling reporter TOPeGFP transgenic mice and found TOPeGFP activation predominantly in proliferating Sox2(+) OE basal cells during early postnatal development. FACS-isolated TOPeGFP(+) OE basal cells are required, but are not sufficient, for formation of spheres. Wnt3a significantly promotes the proliferation of the Sox2(+) OE sphere cells. Wnt-stimulated OE sphere cells maintain their multipotency and can differentiate into most types of neuronal and non-neuronal epithelial cells. Also, Wnt activators shift the production of differentiated cells toward olfactory sensory neurons. Moreover, TOPeGFP(+) cells are robustly increased in the adult OE after injury. In vivo administration of Wnt modulators significantly alters the regeneration potential. This study demonstrates the role of the canonical Wnt signaling pathway in the regulation of OE stem cells or progenitors during development and regeneration.

HSCARG inhibits activation of NF-kappaB by interacting with IkappaB kinase-beta.

HSCARG is a recently identified human NADPH sensor. Our previous studies have shown that HSCARG can affect NO production and cell viability, but the signal pathway mediated by this protein is unknown. Here, we show that HSCARG is involved in the NF-kappaB signaling pathway and find that HSCARG suppresses TNF- and IL1-induced NF-kappaB activation in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence analyses demonstrate that HSCARG interacts and colocalizes with IKKbeta. HSCARG inhibits the phosphorylation of IKKbeta and further blocks the degradation of IkappaBalpha, the substrate of IKKbeta, which retains NF-kappaB in the cytoplasm and suppresses its activity. In addition, our data indicate that IKKbeta is required for HSCARG-inhibited NF-kappaB activation. Our findings delineate a pathway by which HSCARG negatively regulates NF-kappaB activation.

Lrp6-mediated canonical Wnt signaling is required for lip formation and fusion.

Neither the mechanisms that govern lip morphogenesis nor the cause of cleft lip are well understood. We report that genetic inactivation of Lrp6, a co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft lip with cleft palate. The activity of a Wnt signaling reporter is blocked in the orofacial primordia by Lrp6 deletion in mice. The morphological dynamic that is required for normal lip formation and fusion is disrupted in these mutants. The expression of the homeobox genes Msx1 and Msx2 is dramatically reduced in the mutants, which prevents the outgrowth of orofacial primordia, especially in the fusion site. We further demonstrate that Msx1 and Msx2 (but not their potential regulator Bmp4) are the downstream targets of the Wnt/beta-catenin signaling pathway during lip formation and fusion. By contrast, a ;fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a retinoic acid-synthesizing enzyme is ectopically expressed in the upper lip primordia of Lrp6-deficient embryos, indicating a region-specific role of the Wnt/beta-catenin signaling pathway in repressing retinoic acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is required for lip development by orchestrating two distinctively different morphogenetic movements.

PPAR{gamma} accelerates cellular senescence by inducing p16INK4{alpha} expression in human diploid fibroblasts.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in the inhibition of cell growth by promoting cell-cycle arrest, and PPARgamma activation induces the expression of p16(INK4alpha) (CDKN2A), an important cell-cycle inhibitor that can induce senescence. However, the role of PPARgamma in cellular senescence is unknown. Here, we show that PPARgamma promotes cellular senescence by inducing p16(INK4alpha) expression. We found several indications that PPARgamma accelerates cellular senescence, including enhanced senescence-associated (SA)-beta-galactosidase staining, increased G1 arrest and delayed cell growth in human fibroblasts. Western blotting studies demonstrated that PPARgamma activation can upregulate the expression of p16(INK4alpha). PPARgamma can bind to the p16 promoter and induce its transcription, and, after treatment with a selective PPARgamma agonist, we observed more-robust expression of p16(INK4alpha) in senescent cells than in young cells. In addition, our data indicate that phosphorylation of PPARgamma decreased with increased cell passage. Our results provide a possible molecular mechanism underlying the regulation of cellular senescence.

SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts.

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated beta-galactosidase (SA-beta-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan. Western blot results showed that SIRT1 reduced the expression of p16(INK4A) and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16(INK4A) and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.