A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.The G protein-coupled receptors (GPCRs) 1 form a large family of membrane bound proteins that share a unique structural feature comprising seven transmembrane ␣-helices. These molecules act as receptors for a diverse range of extracellular signaling molecules including small molecules (amino acids and biogenic amines), lipids, small bioactive peptides, and large polypeptides (1). They have been used successfully as drug targets by the pharmaceutical industry for a number of years. Attention has focused on a number of proteins that are known to be GPCRs through structural homology but for which no ligand has been identified: so-called orphan receptors. At the same time as the recent discovery of new GPCRs, there has been a renewed focus on discovering potential novel peptides that may act as endogenous ligands for these receptors.Here, we describe the cloning of a novel human orphan receptor, a class I GPCR with sequence similarity to receptors for the neuropeptide galanin. This receptor was given the name AXOR12 in accordance with its position in a series of receptors identified in our organization. AXOR12 has a high degree of homology to the rat orphan receptor GPR54 (2) (81% amino acid identity), which suggests that these two receptors may be orthologs. To identify a ligand for AXOR12, we expressed this receptor in mammalian cells and screened the transfected cells in a functional assay against a library rich in known and putative peptide transmitters. Although there was no activity in response to galanin, we identified three peptides that acted as low potency agonists of AXOR12. These peptides were all derived from invertebrates and shared a C-terminal LRF-or LRW-amide motif.During the preparation of this article, a search of patent literature revealed the existence of additional high potency agonists with sequence similarities to the surrogate agonists identified from the screen. These peptides were deri...

Motor neuron diseases (MNDs) are a group of neurodegenerative disorders with involvement of upper and/or lower motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), progressive bulbar palsy, and primary lateral sclerosis. Recently, we have mapped a new locus for an atypical form of ALS/MND (atypical amyotrophic lateral sclerosis [ALS8]) at 20q13.3 in a large white Brazilian family. Here, we report the finding of a novel missense mutation in the vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) gene in patients from this family. Subsequently, the same mutation was identified in patients from six additional kindreds but with different clinical courses, such as ALS8, late-onset SMA, and typical severe ALS with rapid progression. Although it was not possible to link all these families, haplotype analysis suggests a founder effect. Members of the vesicle-associated proteins are intracellular membrane proteins that can associate with microtubules and that have been shown to have a function in membrane transport. These data suggest that clinically variable MNDs may be caused by a dysfunction in intracellular membrane trafficking.

A mis-sense point mutation in the human VAPB gene is associated with a familial form of motor neuron disease that has been classified as Amyotrophic Lateral Sclerosis type VIII. Affected individuals suffer from a spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or an atypical slowly progressing form of ALS. Mammals have two homologous VAP genes, vapA and vapB. VAPA and VAPB share 76% similar or identical amino acid residues; both are COOH-terminally anchored membrane proteins enriched on the endoplasmic reticulum. Several functions have been ascribed to VAP proteins including membrane trafficking, cytoskeleton association and membrane docking interactions for cytoplasmic factors. It is shown here that VAPA and VAPB are expressed in tissues throughout the body but at different levels, and that they are present in overlapping but distinct regions of the endoplasmic reticulum. The disease-associated mutation in VAPB, VAPB(P56S), lies within a highly conserved N-terminal region of the protein that shares extensive structural homology with the major sperm protein (MSP) from nematodes. The MSP domain of VAPA and VAPB is found to interact with the ER-localized transcription factor ATF6. Over expression of VAPB or VAPB(P56S) attenuates the activity of ATF6-regulated transcription and the mutant protein VAPB(P56S) appears to be a more potent inhibitor of ATF6 activity. These data indicate that VAP proteins interact directly with components of ER homeostatic and stress signalling systems and may therefore be parts of a previously unidentified regulatory pathway. The mis-function of such regulatory systems may contribute to the pathological mechanisms of degenerative motor neuron disease.

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ObjectiveExaggerated central nervous system (CNS) inflammatory responses to peripheral stressors may be implicated in delirium. This study hypothesised that the IL-1β family is involved in delirium, predicting increased levels of interleukin-1β (IL-1β) and decreased IL-1 receptor antagonist (IL-1ra) in the cerebrospinal fluid (CSF) of elderly patients with acute hip fracture. We also hypothesised that Glial Fibrillary Acidic Protein (GFAP) and interferon-γ (IFN-γ) would be increased, and insulin-like growth factor 1 (IGF-1) would be decreased.MethodsParticipants with acute hip fracture aged > 60 (N = 43) were assessed for delirium before and 3–4 days after surgery. CSF samples were taken at induction of spinal anaesthesia. Enzyme-linked immunosorbent assays (ELISA) were used for protein concentrations.ResultsPrevalent delirium was diagnosed in eight patients and incident delirium in 17 patients. CSF IL-1β was higher in patients with incident delirium compared to never delirium (incident delirium 1.74 pg/ml (1.02–1.74) vs. prevalent 0.84 pg/ml (0.49–1.57) vs. never 0.66 pg/ml (0–1.02), Kruskal–Wallis p = 0.03). CSF:serum IL-1β ratios were higher in delirious than non-delirious patients. CSF IL-1ra was higher in prevalent delirium compared to incident delirium (prevalent delirium 70.75 pg/ml (65.63–73.01) vs. incident 31.06 pg/ml (28.12–35.15) vs. never 33.98 pg/ml (28.71–43.28), Kruskal–Wallis p = 0.04). GFAP was not increased in delirium. IFN-γ and IGF-1 were below the detection limit in CSF.ConclusionThis study provides novel evidence of CNS inflammation involving the IL-1β family in delirium and suggests a rise in CSF IL-1β early in delirium pathogenesis. Future larger CSF studies should examine the role of CNS inflammation in delirium and its sequelae.