Jeffrey K. Harrison

Fractalkine and CX3CR1 regulate hippocampal neurogenesis in adult and aged rats

Microglia have neuroprotective capacities, yet chronic activation can promote neurotoxic inflammation. Neuronal fractalkine (FKN), acting on CX(3)CR1, has been shown to suppress excessive microglia activation. We found that disruption in FKN/CX(3)CR1 signaling in young adult rodents decreased survival and proliferation of neural progenitor cells through IL-1β. Aged rats were found to have decreased levels of hippocampal FKN protein; moreover, interruption of CX(3)CR1 function in these animals did not affect neurogenesis. The age-related loss of FKN could be restored by exogenous FKN reversing the age-related decrease in hippocampal neurogenesis. There were no measureable changes in young animals by the addition of exogenous FKN. The results suggest that FKN/CX(3)CR1 signaling has a regulatory role in modulating hippocampal neurogenesis via mechanisms that involve indirect modification of the niche environment. As elevated neuroinflammation is associated with many age-related neurodegenerative diseases, enhancing FKN/CX(3)CR1 interactions could provide an alternative therapeutic approach to slow age-related neurodegeneration.

Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis

Chemokines are a group of pro-inflammatory peptides that mediate leukocyte migration and activation. Several members of the chemokine family have been shown to be synthesized by cells of the central nervous system (CNS). To begin to address the role of chemokine receptors in CNS physiology, we identified, by molecular cloning techniques, the rat orthologs of the chemokine receptors, CCR2, CCR3, CCR5, and CXCR4. CCR2 and CCR5 expression was detected in rat spleen, lung, kidney, thymus and macrophages; CCR5 mRNA was also detected in rat brain. Primary cultures of rat microglia expressed CCR5 mRNA that was regulated by IFN-gamma, while both cultured astrocytes and microglia were found to contain mRNA for CXCR4 and CX3CR1. Induction of experimental allergic encephalomyelitis (EAE) in the rat was accompanied by increased levels of CCR2, CCR5, CXCR4, and CX3CR1 mRNAs in the lumbar spinal cords of animals displaying clinical signs of the disease. These data identify the rat orthologs of chemokine receptors and demonstrate that brain, spinal cord, and cultured glial cells express chemokine receptors that can be regulated both in vitro and in vivo.

Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus

ABSTRACT Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.

NF‐κB‐dependent fractalkine induction in rat aortic endothelial cells stimulated by IL‐1β, TNF‐α, and LPS

Fractalkine is an endothelial cell‐derived CX3C chemokine that is chemotactic mainly to mononuclear cells. Fractalkine was induced in rat aortic endothelial cells (RAEC) by interleu‐kin‐1β (IL‐1β), tumor necrosis factor α (TNF‐α), and lipopolysaccharide (LPS) transcriptionally and translationally. This induction correlated with increased NF‐κB DNA binding activity as determined by gel mobility shift assay. Supershift assays revealed that the NF‐κB subunits p50 and p65 were responsible for κB binding. Accordingly, we examined the role of NF‐κB in fractalkine induction in RAEC through the use of an adenovirus‐mediated mutant IκB as a specific inhibitor. Delivery of a dominant‐negative form of IκBα in RAEC dramatically reduced the induction of fractalkine by these stimuli, suggesting a role for NF‐κB activation in fractalkine induction. The inhibition of fractalkine expression by two potent NF‐κB inhibitors, sulfasalazine and sanguinarine, further supported the central role of NF‐κB in fractalkine transcription regulation and suggested a novel therapeutic target aimed at modulating leukocyte endothelial cell interaction. J. Leukoc. Biol. 67: 577–584; 2000.

Chemokines and their receptors in neurobiology: perspectives in physiology and homeostasis

Chemokines are a large family of small secreted proteins (8-14 kDa) associated with the trafficking of leukocytes in physiological immunosurveillance as well as inflammatory cell recruitment in different disease processes. A limited repertoire of chemokines and their specific cognate receptors are detectable in cells of the CNS such as microglia, astrocytes and neurons under physiological conditions. Coupled with distinct patterns of ligand and receptor expression in various pathologies including multiple sclerosis, trauma, neuro-AIDS, Alzheimers disease, stroke, neuro- and glioblastomas, such phenomena have fueled the strong belief that chemokines must fulfill significant and potentially diverse functional roles in the CNS.

Cultured rat microglia express functional beta-chemokine receptors.

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3.

The RNA-mediated pathogenesis model for the myotonic dystrophies DM1 and DM2 proposes that mutant transcripts from the affected genes sequester a family of double-stranded RNA-binding factors, the muscleblind proteins MBNL1, MBNL2 and MBNL3, in the nucleus. These proteins are homologues of the Drosophila muscleblind proteins that are required for the terminal differentiation of muscle and photoreceptor tissues, and thus nuclear sequestration of the human proteins might impair their normal function in muscle and eye development and maintenance. To examine this model further, we analyzed the expression pattern of the mouse Mbnl1, Mbnl2, and Mbnl3 genes during embryonic development and compared muscleblind gene expression to Dmpk since the RNA pathogenesis model for DM1 requires the coordinate synthesis of mutant Dmpk transcripts and muscleblind proteins. Our studies reveal a striking overlap between the expression of Dmpk and the muscleblind genes during development of the limbs, nervous system and various muscles, including the diaphragm and tongue.

Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart.

Fractalkine is distinguished structurally from other chemokines in that it contains a mucinlike stalk that tethers a CX3C chemokine module to a transmembrane‐spanning region; its expression in cultured endothelial cells has been shown to be up‐regulated by tumor necrosis factor α (TNF‐α) and interleukin‐1 (IL‐1). The purpose of this study was to determine whether fractalkine is expressed, in a proinflammatory agent‐regulated manner, by cardiac endothelial cells in vivo. Steady state levels of fractalkine mRNA were increased in rat cardiac tissues after in vivo treatment with lipopolysaccharide (LPS), IL‐1, or TNF‐α. In situ hybridization and immunohistochemical analysis revealed that endothelial cells of the coronary vasculature and endocardium were the principal source of proinflammatory agent‐inducible fractalkine, although some fractalkine immunoreactivity was also found on the myocytes. These data are the first demonstration of in vivo cardiac endothelial cell fractalkine expression and regulation by proinflammatory agents such as LPS, IL‐1, or TNF‐α. Cardiac endothelial cell‐expressed fractalkine may contribute to the influx of leukocytes into the heart during inflammation. J. Leukoc. Biol. 66: 937–944; 1999.

Chemokines and Alzheimer’s disease

In recent years, increasing attention has been focused on chemokines as inflammatory mediators in the CNS. The limited number of studies that have investigated chemokine and chemokine receptor expression in Alzheimers disease (AD) brain and in cell culture models seem to support a role for inflammation in AD pathogenesis. Here we provide a review of these studies, but in addition, point out the possible role of chemokines as communication molecules between neurons and microglia. Understanding neuron-microglia interactions is essential for understanding AD pathogenesis, and disturbances in chemokine-mediated intercellular communication may contribute toward a generalized impairment of microglial cell function.

Identification of Two Rat Genes Orthologous to the Human Interleukin-8 Receptors

The genes encoding two functional human interleukin-8 (IL-8) receptors have been identified by molecular cloning techniques and they are members of the rhodopsin G-protein coupled receptor (GCR) superfamily. We report the molecular cloning of two rat GCR genes (rat CXCR1-like and rat CXCR2) whose conceptualized amino acid sequences are approximately 70% identical to the human IL-8 A and B receptor subtypes. The murine GRO-like peptide, macrophage inflammatory peptide-2 (MIP-2), elevates intracellular calcium levels in HEK293 cells expressing the rat CXCR2 receptor. Southern blot analysis of restriction-digested rodent and human genomic DNAs indicate that rat CXCR1-like and CXCR2 are: 1) each single copy genes in the rat genome; 2) most closely related to the human IL-8 receptor genes; and 3) orthologous to two previously identified murine genes. CXCR2 mRNA is detected in adult rat lung, spleen, and neutrophils. CXCR1-like mRNA can be detected in adult rat lung, native rat macrophages, and a rat alveolar macrophage cell line (NR8383). These data identify the rat orthologs of the human IL-8 receptors, and describe cellular and tissue targets of rat C-X-C chemokine peptides.