Absence of Receptor for Advanced Glycation End Product (RAGE) Reduces Inflammation and Extends Survival in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signalling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE and compared them with SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signalling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by ~ 3 weeks and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS and highlights RAGE as a potential immune therapeutic target for ALS.

Pharmacological inhibition of complement C5a-C5a1 receptor signalling ameliorates disease pathology in the hSOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease without effective treatment. The complement system is up-regulated in ALS, with recent studies indicating that the activation product C5a accelerates disease progression via the C5a1 receptor (C5aR1). We therefore examined the therapeutic effect of C5a1 receptor antagonism in hSOD1G93A mice, the most widely used preclinical model of ALS. EXPERIMENTAL APPROACH: The selective and orally active C5a1 receptor antagonist, PMX205, was administered to hSOD1G93A mice in drinking water, both pre- and post-disease onset. Blood, brain and spinal cord pharmacokinetics were performed using LC-MS/MS methods. Effects of PMX205 on hSOD1G93A disease progression was determined using body weight, hindlimb grip strength, survival time and blood analysis. KEY RESULTS: PMX205 entered the intact CNS at pharmacologically active concentrations, with increased entry observed in hSOD1G93A mice as the disease progressed, in line with augmented blood-brain barrier breakdown. hSOD1G93A mice treated with PMX205 before disease onset had significantly improved hindlimb grip strength, slower disease progression and extended survival, compared with vehicle treatment. These improvements were associated with reductions in pro-inflammatory monocytes and granulocytes and increases in T-helper lymphocytes in peripheral blood. PMX205 treatment beginning 3 weeks following disease onset also attenuated disease progression, significantly extending survival. CONCLUSION AND IMPLICATIONS: These results confirm that C5a1 receptors play a pathogenic role in hSOD1G93A mice, further validating the C5a-C5a1 receptor signalling axis as a potential therapeutic target to slow disease progression in ALS.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Dysregulation of the complement cascade in the hSOD1G93A transgenic mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Components of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS); however, a comprehensive examination of complement expression in this disease has not been performed. This study therefore aimed to determine the expression of complement components (C1qB, C4, factor B, C3/C3b, C5 and CD88) and regulators (CD55 and CD59a) in the lumbar spinal cord of hSOD1(G93A) mice during defined disease stages. METHODS: hSOD1(G93A) and wild-type mice were examined at four different ages of disease progression. mRNA and protein expression of complement components and regulators were examined using quantitative PCR, western blotting and ELISA. Localisation of complement components within lumbar spinal cord was investigated using immunohistochemistry. Statistical differences between hSOD1(G93A) and wild-type mice were analysed using a two-tailed t-test at each stage of disease progression. RESULTS: We found several early complement factors increased as disease progressed, whilst complement regulators decreased; suggesting overall increased complement activation through the classical or alternative pathways in hSOD1(G93A) mice. CD88 was also increased during disease progression, with immunolocalisation demonstrating expression on motor neurons and increasing expression on microglia surrounding the regions of motor neuron death. CONCLUSIONS: These results indicate that local complement activation and increased expression of CD88 may contribute to motor neuron death and ALS pathology in the hSOD1(G93A) mouse. Hence, reducing complement-induced inflammation could be an important therapeutic strategy to treat ALS.

Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

The molecular mechanisms involved in non­small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo.

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Expression and in vitro functions of the ghrelin axis in endometrial cancer.

Ghrelin is a peptide hormone produced in the stomach and a range of other tissues, where it has endocrine, paracrine and autocrine roles in both normal and disease states. Ghrelin has been shown to be an important growth factor for a number of tumours, including prostate and breast cancers. In this study, we examined the expression of the ghrelin axis (ghrelin and its receptor, the growth hormone secretagogue receptor, GHSR) in endometrial cancer. Ghrelin is expressed in a range of endometrial cancer tissues, while its cognate receptor, GHSR1a, is expressed in a small subset of normal and cancer tissues. Low to moderately invasive endometrial cancer cell lines were examined by RT-PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa, HEC1B and KLE endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis and represent a novel treatment target.

Maternal dietary zinc supplementation prevents aberrant behaviour in an object recognition task in mice offspring exposed to LPS in early pregnancy.

Maternal infection during pregnancy is associated with an increased risk of neurodevelopmental damage. While the mechanism is unclear accumulating evidence suggests that the maternal inflammatory response may be responsible. Metallothionein (MT) is a zinc (Zn)-binding protein that when induced in the mother's liver during the acute phase response has been found to cause a fetal Zn deficiency. Infection-mediated fetal Zn deficiency in early pregnancy has been shown to cause teratogenicity which can be prevented by dietary Zn supplementation throughout pregnancy. This study examined whether cognitive impairments can be caused by lipopolysaccharide (LPS) administration early in pregnancy and whether dietary Zn supplementation can ameliorate these changes. Maternal inflammation induced by LPS at gestation day (GD) 8 did not affect spatial learning or memory of adult mice offspring in a water cross-maze escape task. However, in an object recognition task, where control mice demonstrated good visual recognition memory by exploring a novel object more than a familiar object, LPS-treated offspring demonstrated abnormal perseverant exploration towards the familiar object that cannot be explained in full by impaired object recognition memory. In comparison, offspring of mice from dams given LPS and dietary Zn supplementation displayed normal object recognition task performance. Microarray analysis on the brain of GD 12 fetuses did not identify any differentially expressed genes between treatment groups. This study demonstrates that LPS administration in early pregnancy can cause an anomaly in object recognition that can be measured in adult offspring. This aberrant behaviour can be prevented by dietary Zn supplementation during pregnancy, thus providing a nutritional strategy to limit neurodevelopmental damage caused by infections early in pregnancy.