Robin J. Parks

The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response

The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-κB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1β in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.

Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors.

Previous studies using adenoviral (Ad) vectors expressing human alpha1-antitrypsin (hAAT) under the control of ubiquitous promoters (RSV, mPGK) elicited the production of antibodies to hAAT in some mouse strains (C3H/HeJ and BALB/c) but not in others (C57BL/6J). In contrast, when a helper-dependent Ad vector (AdSTK109) with all viral coding sequences deleted and expressing hAAT from human genomic DNA with the endogenous promoter was used, C3H/HeJ mice failed to develop antibodies and demonstrated long-term expression. These results suggested that promoter choice and/or properties of the vector itself might influence the host immune response to the transgene product. Direct comparison of first-generation vectors expressing the hAAT cDNA from a ubiquitous mouse PGK promoter rather than from a liver-specific mouse albumin promoter demonstrated that an antibody response to hAAT occurred with the mPGK promoter but not with the albumin promoter in C3H/HeJ mice. As expected, neither vector elicits an antibody response in C57BL/6J mice. Coinjection of the two first-generation vectors containing the mPGK and albumin promoter in C3H/HeJ mice induced an antibody response with resulting loss of detectable hAAT from the sera of the injected mice in 3-4 weeks. From these data, we conclude that under certain conditions, the choice of promoter with its associated liver-specific expression can modulate the host immune response to the transgene independent of viral backbone.

Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation

To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.

Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies

Although the molecular defect causing Duchenne/Becker muscular dystrophy (DMD/BMD) was identified nearly 20 years ago, the development of effective therapeutic strategies has nonetheless remained a daunting challenge. Over the years, a variety of different approaches have been explored in an effort to compensate for the lack of the DMD gene product called dystrophin. This review not only presents some of the most promising molecular, cellular, and pharmacological strategies but also highlights some issues that need to be addressed before considering their implementation. Specifically, we describe current strategies being developed to exogenously deliver healthy copies of the dystrophin gene to dystrophic muscles. We present the findings of several studies that have focused on repairing the mutant dystrophin gene using various approaches. We include a discussion of cell‐based therapies that capitalize on the use of myoblast or stem cell transfer. Finally, we summarize the results of several studies that may eventually lead to the development of appropriate drug‐based therapies. In this context, we review our current knowledge of the mechanisms regulating expression of utrophin, the autosomal homologue of dystrophin. Given the complexity associated with the dystrophic phenotype, it appears likely that a combinatorial approach involving different therapeutic strategies will be necessary for the appropriate management and eventual treatment of this devastating neuromuscular disease. Chakkalakal J. V., Thompson J., Parks R. J., Jasmin B. J. Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies. FASEB J. 19, 880–891 (2005)

An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method

The two-plasmid rescue method of constructing Ad vectors, which relies on either homologous or Cre-mediated recombination between two plasmids cotransfected into 293 or 293Cre4 cells, respectively, offers advantages over other approaches because of its simplicity. We have improved the efficiency of vector construction by both homologous and Cre-mediated recombination by replacing the single ITR in the shuttle plasmid with a head-to-head ITR junction. We have also expanded the versatility of this method by incorporating a Cre expression cassette into the plasmids to permit high-efficiency Cre-mediated vector rescue using 293 cells, abrogating the need for Cre-expressing cell lines. This new system retains the simplicity of the original but results in an approximately 100-fold increase in the number of recombinant viruses produced, all of which contain the foreign DNA insert, and allows high-efficiency Cre-mediated vector isolation using any E1-complementing cell line.

A Unique Interplay Between Rap1 and E-Cadherin in the Endocytic Pathway Regulates Self-Renewal of Human Embryonic Stem Cells† ‡ §

Regulatory mechanisms pertaining to the self‐renewal of stem cells remain incompletely understood. Here, we show that functional interactions between small GTPase Rap1 and the adhesion molecule E‐cadherin uniquely regulate the self‐renewal of human embryonic stem cells (hESCs). Inhibition of Rap1 suppresses colony formation and self‐renewal of hESCs, whereas overexpression of Rap1 augments hESC clonogenicity. Rap1 does not directly influence the expression of the pluripotency genes Oct4 and Nanog. Instead, it affects the endocytic recycling pathway involved in the formation and maintenance of E‐cadherin‐mediated cell–cell cohesion, which is essential for the colony formation and self‐renewal of hESCs. Conversely, distinct from epithelial cells, disruption of E‐cadherin mediated cell–cell adhesions induces lysosome delivery and degradation of Rap1. This in turn leads to a further downregulation of E‐cadherin function and a subsequent reduction in hESC clonogenic capacity. These findings provide the first demonstration that the interplay between Rap1 and E‐cadherin along the endocytic recycling pathway serves as a timely and efficient mechanism to regulate hESC self‐renewal. Given the availability of specific activators for Rap1, this work provides a new perspective to enable better maintenance of human pluripotent stem cells. STEM CELLS 2010;28:247–257

Missense Mutation in APOC3 within the C-terminal Lipid Binding Domain of Human ApoC-III Results in Impaired Assembly and Secretion of Triacylglycerol-rich Very Low Density Lipoproteins EVIDENCE THAT ApoC-III PLAYS A MAJOR ROLE IN THE FORMATION OF LIPID PRECURSORS WITHIN THE MICROSOMAL LUMEN

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL1, the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys58 with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys58 and Lys60 with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKzeta initiates RhoGDI release and Rac1 activation. In DGKzeta-deficient fibroblasts PAK1 phosphorylation and Rac1-RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKzeta, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKzeta stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKzeta, and PA to the activation of Rac1: the PA generated by DGKzeta activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.

Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase ζ

ABSTRACT Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase ζ (DGK-ζ), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-ζ and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-ζ in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-ζ kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-ζ mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-ζ promotes neurite outgrowth through association with the GTPase Rac1. DGK-ζ colocalized with Rac1 in neuronal processes and DGK-ζ-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-ζ directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-ζ mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-ζ with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-ζ binding to active Rac1. Collectively, these findings suggest DGK-ζ, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.

Plasma butyrylcholinesterase regulates ghrelin to control aggression

Significance Butyrylcholinesterase (BChE), a common plasma enzyme, has been known for decades but its real physiological roles are just beginning to emerge. Although BChE eliminates the neurotransmitter acetylcholine, it is not vital for locomotion, cognition, or other cholinergic functions. Nevertheless, we now find that circulating BChE has a large impact on aggressive behavior in mice that is attributable to its ability to inactivate ghrelin, a peptide hormone involved in hunger, feeding, and stress. A key observation was decreased fighting among group-housed male mice overexpressing BChE after viral gene transfer. In contrast, BChE knockout mice exhibited increased fighting. These effects mirrored changes in plasma levels of active ghrelin. Controlling them might offer therapeutic potential for certain behavioral disorders. Ongoing mouse studies of a proposed therapy for cocaine abuse based on viral gene transfer of butyrylcholinesterase (BChE) mutated for accelerated cocaine hydrolysis have yielded surprising effects on aggression. Further investigation has linked these effects to a reduction in circulating ghrelin, driven by BChE at levels ∼100-fold above normal. Tests with human BChE showed ready ghrelin hydrolysis at physiologic concentrations, and multiple low-mass molecular dynamics simulations revealed that ghrelin’s first five residues fit sterically and electrostatically into BChE’s active site. Consistent with in vitro results, male BALB/c mice with high plasma BChE after gene transfer exhibited sharply reduced plasma ghrelin. Unexpectedly, such animals fought less, both spontaneously and in a resident/intruder provocation model. One mutant BChE was found to be deficient in ghrelin hydrolysis. BALB/c mice transduced with this variant retained normal plasma ghrelin levels and did not differ from untreated controls in the aggression model. In contrast, C57BL/6 mice with BChE gene deletion exhibited increased ghrelin and fought more readily than wild-type animals. Collectively, these findings indicate that BChE-catalyzed ghrelin hydrolysis influences mouse aggression and social stress, with potential implications for humans.