Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

Primary cilia are small microtubule‐based organelles capable of transducing signals from growth factor receptors embedded in the cilia membrane. Developmentally, oligodendrocyte progenitor cells (OPCs) express genes associated with primary cilia assembly, disassembly, and signaling, however, the importance of primary cilia for adult myelination has not been explored. We show that OPCs are ciliated in vitro and in vivo, and that they disassemble their primary cilia as they progress through the cell cycle. OPC primary cilia are also disassembled as OPCs differentiate into oligodendrocytes. When kinesin family member 3a (Kif3a), a gene critical for primary cilium assembly, was conditionally deleted from adult OPCs in vivo (Pdgfrα‐CreER™:: Kif3a fl/fl transgenic mice), OPCs failed to assemble primary cilia. Kif3a‐deletion was also associated with reduced OPC proliferation and oligodendrogenesis in the corpus callosum and motor cortex and a progressive impairment of fine motor coordination.

Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides (''CpG'' motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor ␣. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colonystimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism. J. Leukoc. Biol. 66: 542-548; 1999.

Throughout life, oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes. OPCs express cell surface receptors and channels that allow them to detect and respond to neuronal activity, including voltage‐gated calcium channel (VGCC)s. The major L‐type VGCC expressed by developmental OPCs, CaV1.2, regulates their differentiation. However, it is unclear whether CaV1.2 similarly influences OPC behavior in the healthy adult central nervous system (CNS). To examine the role of CaV1.2 in adulthood, we conditionally deleted this channel from OPCs by administering tamoxifen to P60 Cacna1c fl/fl (control) and Pdgfrα‐CreER:: Cacna1c fl/fl (CaV1.2‐deleted) mice. Whole cell patch clamp analysis revealed that CaV1.2 deletion reduced L‐type voltage‐gated calcium entry into adult OPCs by ~60%, confirming that it remains the major L‐type VGCC expressed by OPCs in adulthood. The conditional deletion of CaV1.2 from adult OPCs significantly increased their proliferation but did not affect the number of new oligodendrocytes produced or influence the length or number of internodes they elaborated. Unexpectedly, CaV1.2 deletion resulted in the dramatic loss of OPCs from the corpus callosum, such that 7 days after tamoxifen administration CaV1.2‐deleted mice had an OPC density ~42% that of control mice. OPC density recovered within 2 weeks of CaV1.2 deletion, as the lost OPCs were replaced by surviving CaV1.2‐deleted OPCs. As OPC density was not affected in the motor cortex or spinal cord, we conclude that calcium entry through CaV1.2 is a critical survival signal for a subpopulation of callosal OPCs but not for all OPCs in the mature CNS.

It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The dogma of an exclusively retinal distribution has arisen because Northern blot analyses have previously shown only modest hybridisation in non-retinal tissues. Others have re-interpreted this as indicating that EAAT5 was only present in retinal tissues. However, this view appears to be erroneous; recent evidence demonstrating abundant expression of EAAT5 in rat testis prompted us to re-examine this dogma. A new antibody was developed to an intracellular loop region of rat EAAT5. This new tool, in concert with RT-PCR and sequencing, demonstrated that EAAT5 is widely distributed at the mRNA and protein levels in many non-nervous tissues including liver, kidney, intestine, heart, lung, and skeletal muscle. We conclude that EAAT5 is a widely distributed protein. Whether it functions in all locations as a glutamate transporter, or mainly as a glutamate-gated chloride conductance, remains to be determined.