Enantiomeric Agonists of the Type 2 Cannabinoid Receptor Reduce Retinal Damage during Proliferative Vitreoretinopathy and Inhibit Hyperactive Microglia In Vitro.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, ß-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both ß-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

Ectopic expression of aPKC-mediated phosphorylation in p300 modulates hippocampal neurogenesis, CREB binding and fear memory differently with age.

Epigenetic modifications have become an emerging interface that links extrinsic signals to alterations of gene expression that determine cell identity and function. However, direct signaling that regulates epigenetic modifications is unknown. Our previous work demonstrated that phosphorylation of CBP at Ser 436 by atypical protein kinase C (aPKC) regulates age-dependent hippocampal neurogenesis and memory. p300, a close family member of CBP, lacks the aPKC-mediated phosphorylation found in CBP. Here, we use a phosphorylation-competent p300 (G442S) knock-in (KI) mouse model that ectopically expresses p300 phosphorylation in a homologous site to CBP Ser436, and assess its roles in modulating hippocampal neurogenesis, CREB binding ability, and fear memory. Young adult (3 months) p300G422S-KI mice exhibit enhanced hippocampal neurogenesis due to increased cell survival of newly-generated neurons, without alterations in CREB binding and contextual fear memory. On the other hand, mature adult (6 months) p300G422S-KI mice display reduced CREB binding, associated with impaired contextual fear memory without alterations in hippocampal neurogenesis. Additionally, we show that repulsive interaction between pS133-CREB and pS422-p300G422S may contribute to the reduced CREB binding to p300G422S. Together, these data suggest that a single phosphorylation change in p300 has the capability to modulate hippocampal neurogenesis, CREB binding, and associative fear memory.

The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner.

While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A) in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.

Vascular Smooth Muscle-Specific EP4 Receptor Deletion in Mice Exacerbates Angiotensin II-Induced Renal Injury.

AIMS: Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs is contraindicated in hypertension, as it may reduce glomerular filtration rate (GFR) and renal blood flow. However, the identity of the specific eicosanoid and receptor underlying these effects is not known. We hypothesized that vascular smooth muscle prostaglandin E2 (PGE2) E-prostanoid 4 (EP4) receptor deletion predisposes to renal injury via unchecked vasoconstrictive actions of angiotensin II (AngII) in a hypertension model. Mice with inducible vascular smooth muscle cell (VSMC)-specific EP4 receptor deletion were generated and subjected to AngII-induced hypertension. RESULTS: EP4 deletion was verified by PCR of aorta and renal vessels, as well as functionally by loss of PGE2-mediated mesenteric artery relaxation. Both AngII-treated groups became similarly hypertensive, whereas albuminuria, foot process effacement, and renal hypertrophy were exacerbated in AngII-treated EP4VSMC-/- but not in EP4VSMC+/+ mice and were associated with glomerular scarring, tubulointerstitial injury, and reduced GFR. AngII-treated EP4VSMC-/- mice exhibited capillary damage and reduced renal perfusion as measured by fluorescent bead microangiography and magnetic resonance imaging, respectively. NADPH oxidase 2 (Nox2) expression was significantly elevated in AngII-treated EP4-/- mice. EP4-receptor silencing in primary VSMCs abolished PGE2 inhibition of AngII-induced Nox2 mRNA and superoxide production. INNOVATION: These data suggest that vascular EP4 receptors buffer the actions of AngII on renal hemodynamics and oxidative injury. CONCLUSION: EP4 agonists may, therefore, protect against hypertension-associated kidney damage. Antioxid. Redox Signal. 25, 642-656.

Metformin Acts on Two Different Molecular Pathways to Enhance Adult Neural Precursor Proliferation/Self-Renewal and Differentiation.

The recruitment of endogenous adult neural stem cells for brain repair is a promising regenerative therapeutic strategy. This strategy involves stimulation of multiple stages of adult neural stem cell development, including proliferation, self-renewal, and differentiation. Currently, there is a lack of a single therapeutic approach that can act on these multiple stages of adult neural stem cell development to enhance neural regeneration. Here we show that metformin, an FDA-approved diabetes drug, promotes proliferation, self-renewal, and differentiation of adult neural precursors (NPCs). Specifically, we show that metformin enhances adult NPC proliferation and self-renewal dependent upon the p53 family member and transcription factor TAp73, while it promotes neuronal differentiation of these cells by activating the AMPK-aPKC-CBP pathway. Thus, metformin represents an optimal candidate neuro-regenerative agent that is capable of not only expanding the adult NPC population but also subsequently driving them toward neuronal differentiation by activating two distinct molecular pathways.

The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity.

To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

Functional analysis of the impact of ORMDL3 expression on inflammation and activation of the unfolded protein response in human airway epithelial cells.

BACKGROUND: The gene ORMDL3 was shown to be associated with early-onset asthma susceptibility in multiple independent genome-wide and candidate-gene association studies. Asthmatic patients have elevated expression levels of this gene. ORMDL3 encodes a transmembrane protein localized in the endoplasmic reticulum (ER) that may be involved in ER stress and inflammation. It is essential to validate the genetic associations linking ORMDL3 with asthma through functional studies that confirm the biological relevance of this gene in disease. We investigated the effects of manipulating ORMDL3 expression levels in vitro in airway cells on innate immune inflammatory responses, ER stress and activation of the unfolded protein response (UPR). METHODS: ORMDL3 expression levels were manipulated in airway cells using an overexpression plasmid and siRNA technologies. Successful modulation of ORMDL3 was confirmed at both the gene and protein level. The functional impact of modulation of ORMDL3 expression levels on inflammatory responses and activation of the UPR were quantified using complementary cellular and molecular immunology techniques. RESULTS: Cells with altered ORMDL3 levels responded equally well to innate immune stimuli and produced similar levels of pro-inflammatory cytokines compared to wild-type cells. Treatment with ER stress inducers, thapsigargin and tunicamycin, resulted in activation of the unfolded protein response (UPR). However, we observed no difference in UPR activation in cells with ORMDL3 knockdown compared to cells with normal ORMDL3 levels. CONCLUSIONS: Our results suggest that ORMDL3 variation in the airway epithelium is unlikely to play a significant role in modulating innate immune responses and the UPR in the lung.

Atypical activation of the unfolded protein response in cystic fibrosis airway cells contributes to p38 MAPK-mediated innate immune responses.

Inflammatory lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF); understanding what produces dysregulated innate immune responses in CF cells will be pivotal in guiding the development of novel anti-inflammatory therapies. To elucidate the molecular mechanisms that mediate exaggerated inflammation in CF following TLR signaling, we profiled global gene expression in immortalized human CF and non-CF airway cells at baseline and after microbial stimulation. Using complementary analysis methods, we observed a signature of increased stress levels in CF cells, specifically characterized by endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and MAPK signaling. Analysis of ER stress responses revealed an atypical induction of the UPR, characterized by the lack of induction of the PERK-eIF2α pathway in three complementary model systems: immortalized CF airway cells, fresh CF blood cells, and CF lung tissue. This atypical pattern of UPR activation was associated with the hyperinflammatory phenotype in CF cells, as deliberate induction of the PERK-eIF2α pathway with salubrinal attenuated the inflammatory response to both flagellin and Pseudomonas aeruginosa. IL-6 production triggered by ER stress and microbial stimulation were both dependent on p38 MAPK activity, suggesting a molecular link between both signaling events. These data indicate that atypical UPR activation fails to resolve the ER stress in CF and sensitizes the innate immune system to respond more vigorously to microbial challenge. Strategies to restore ER homeostasis and normalize the UPR activation profile may represent a novel therapeutic approach to minimize lung-damaging inflammation in CF.

Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death, and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3 was. PCV1ORF3-associated cell death was caspase dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase 1 (PARP) cleavage; however, whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids contain a domain capable of inducing cell death, whereas the C terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not determined or not solely determined by the ORF3 protein.