Shannon J. Beasley

Phosphorothioate backbone modification modulates macrophage activation by CpG DNA.

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.

The actions of bacterial DNA on murine macrophages.

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 a. 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 colony‐stimulating 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.

Excitatory amino acid transporter 5 is widely expressed in peripheral tissues.

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.

A new splice variant of the glutamate–aspartate transporter: Cloning and immunolocalization of GLAST1c in rat, pig and human brains

GLAST (EAAT1) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles in terminating excitatory transmission in grey matter, as well as pathophysiological roles, including protecting white matter from excitotoxic injury. In normal brain, alternative splicing of GLAST has been described: GLAST1a and GLAST1b arise from the splicing out of exons 3 and 9, respectively. This study describes the isolation of a novel cDNA clone from neonatal hypoxic pig brain, referred to as GLAST1c, where exons 5 and 6 are skipped. GLAST1c encodes a protein of 430 amino acids. RT-PCR analysis showed that GLAST1c mRNA was readily detectable in control and hypoxic pig cortex, as well as in various brain regions of rat (cortex, mid, hind and cerebellum), and human cortex, retina and optic nerve. We have raised antibodies that selectively recognize GLAST1c and demonstrate expression of this novel splice variant in astrocytes and oligodendrocytes in rat brain, pig brain and human brain, including grey and white matter. Similarly expression of GLAST1c was observed in primary astrocyte cultures and in cultured oligodendrocytes. In unstimulated astrocytes GLAST1c exhibited an intracellular peri-nuclear distribution similar to that observed when GFP-tagged GLAST1c was transfected into COS 7 cells. In astrocytes this protein rapidly redistributed to the surface upon stimulation of protein kinase with phorbol esters. We conclude that GLAST1c may represent an astrocyte and oligodendrocyte glutamate transporter, though this could not be formally validated by D-aspartate uptake studies, due to the low transfection efficiency of constructs into COS 7 cells.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-γ

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour‐specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B‐CLL might use an escape strategy, the current studies compared B‐CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B‐CLL cells in response to IFN‐γ was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B‐CLL cells upregulated TAP protein expression in response to IFN‐γ. Responsiveness of B‐CLL MHC class I mRNA to IFN‐γ was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B‐CLL than normal B cells, as a result of the described release of β2m and β2m‐free class I heavy chains from the membrane. This relative MHC class I expression defect of B‐CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Autoantibodies and their potential roles in diseases of the nervous system

Autoantibodies are found in many disorders of the nervous system, including diseases affecting the peripheral nervous system (e.g. Guillain–Barré syndrome and other peripheral neuropathies), the central nervous system (e.g. neuromyelitis optica, multiple sclerosis and limbic encephalitis) and the neuromuscular junction (e.g. myasthenia gravis), as well as in neuropsychiatric disorders and in paraneoplastic syndromes. Some of these antibodies are likely to play an important pathogenic role in disease development, whereas others might be useful biomarkers for disease. In the present review, the features of an autoantibody that enable it to be pathogenic (e.g. the antibody isotype and the target antigen against which the antibody is directed) and some of the different mechanisms by which pathogenic autoantibodies could act will be discussed. These mechanisms include antibody‐mediated cell lysis, opsonization of target proteins for attack by macrophages, cross‐linking of Fc receptors, blocking or destroying receptors involved in neurotransmission or cellular homeostasis, and blocking of repair mechanisms such as remyelination. Examples will be given of diseases where these antibody‐mediated mechanisms are known or thought to be active. Finally, approaches to treating autoantibody‐mediated disease in the nervous system will be briefly reviewed.

Development of a simple in vitro mature mixed glial model to investigate differential expression of cell markers on glial cells and their potential as targets in multiple sclerosis

...............................................................................................i Declaration by author..............................................................................iv Publications during candidature................................................................v Publications included in thesis..................................................................v Contributions by others to the thesis...........................................................vi Statement of parts of the thesis submitted to qualify for the award of another degree.................................................................................................vi Acknowledgements................................................................................vii

Expression of glutamate transporter splice variants on oligodendrocytes in rat brain and in mixed glial cultures

This free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, AustraliaThis free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, Australia

Glutamate neurotoxicity, transport and alternate splicing of transporters

Glutamate is the major excitatory neurotransmitter in the central nervous system and its levels in the synaptic cleft are tightly controlled by high affinity glutamate transporters (also known as Excitatory Amino Acid Transporters or EAATs). The EAAT family is comprised of five members (EAAT1-5), and these transporters are subject to alternative splicing. Alternative splicing of the EAAT genes is a fundamental mechanism that can give rise to multiple distinct mRNA transcripts, producing protein isoforms with potentially altered functions. Numerous splice variants of EAATs have been identified in humans, rodents, and other mammalian species. All splice variants of EAATs cloned to date are either exon-skipping and/or intron-retaining types. These modifications may impact upon protein structure, posttranslational modification, function, cellular localization, and trafficking. Message and protein for these splice variants are detectable in the normal brain and, in many instances, have been shown to be induced by pathophysiological insults such as hypoxia. In addition, aberrant expression of EAAT splice variants has been reported in neurodegenerative conditions such as amyotrophic lateral sclerosis, Alzheimers disease, ischemic stroke, and age- related macular degeneration. These EAAT variants may represent therapeutic targets and thus require an improved understanding of their regulation. This chapter describes recent developments in investigating the molecular heterogeneity, localization, function, structure, and regulation of the EAATs and their splice variants.