Yvonne J. Hort

Critical role for GALR1 galanin receptor in galanin regulation of neuroendocrine function and seizure activity

The GALR1 galanin receptor is expressed at high levels within the central nervous system. To determine which specific actions of galanin are mediated by GALR1, we have developed mice with an insertional inactivating mutation within the gene encoding GALR1 (Galr1). Homozygous Galr1-/- mice are viable and capable of breeding. They exhibit no significant difference in growth rate relative to Galr1+/+ controls but have reduced circulating levels of insulin-like growth factor-I (IGF-I) and exhibit spontaneous tonic-clonic seizures. The phenotype of these mice identifies a critical role for GALR1 in neuroendocrine regulation and in mediating the anti-seizure activity of galanin.

Critical role for Y1 receptors in mesenchymal progenitor cell differentiation and osteoblast activity.

The neuropeptide Y (NPY) system has been implicated in the regulation of bone homeostasis and osteoblast activity, but the mechanism behind this is unclear. Here we show that Y1 receptor signaling is directly involved in the differentiation of mesenchymal progenitor cells isolated from bone tissue, as well as the activity of mature osteoblasts. Importantly, the mRNA levels of two key osteogenic transcription factors, runx2 and osterix, as well as the adipogenic transcription factor PPAR‐γ, were increased in long bones of Y1−/− mice compared with wild‐type mice. In vitro, bone marrow stromal cells (BMSCs) isolated from Y1−/− mice formed a greater number of mineralized nodules under osteogenic conditions and a greater number of adipocytes under adipogenic conditions than controls. In addition, both the number and size of fibroblast colony‐forming units formed in vitro by purified osteoprogenitor cells were increased in the absence of the Y1 receptors, suggestive of enhanced proliferation and osteogenesis. Furthermore, the ability of two specific populations of mesenchymal progenitor cells isolated from bone tissue, an immature mesenchymal stem cell population and a more committed osteoprogenitor cell population, to differentiate into osteoblasts and adipocytes in vitro was enhanced in the absence of Y1 receptor signaling. Finally, Y1 receptor deletion also enhanced the mineral‐producing ability of mature osteoblasts, as shown by increased in vitro mineralization by BMSCs isolated from osteoblast‐specific Y1−/− mice. Together these data demonstrate that the NPY system, via the Y1 receptor, directly inhibits the differentiation of mesenchymal progenitor cells as well as the activity of mature osteoblasts, constituting a likely mechanism for the high‐bone‐mass phenotype evident in Y1−/− mice.

Gene duplication of the human peptide YY gene (PYY) generated the pancreatic polypeptide gene (PPY) on chromosome 17q21.1

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are structurally related but functionally diverse peptides, encoded by separate genes and expressed in different tissues. Although the human NPY gene has been mapped to chromosome 7, we demonstrate here that the genes for human PYY and PP (PPY) are localized only 10 kb apart from each other on chromosome 17q21.1. The high degree of homology between the members of this gene family, both in primary sequence and exon/intron structure, suggests that the NPY and the PYY genes arose from an initial gene duplication event, with a subsequent tandem duplication of the PYY gene being responsible for duplication of the PYY gene being responsible for the creation of the PPY gene. A second weaker hybridization signal also found on chromosome 17q11 and results obtained by Southern blot analysis suggest that the entire PYY-PPY region has undergone a further duplication event.

Differentiation of adult mouse olfactory precursor cells into hair cells in vitro.

Many forms of deafness result from degeneration of the sensory cells for hearing, the hair cells in the cochlea. Stem cells offer a potential cell‐based therapy for the treatment of deafness. Here, we investigate whether adult olfactory precursor cells can differentiate into hair cells in culture. Precursor cells were isolated from mouse olfactory neuroepithelium, were sphere‐forming, showed proliferative capacity, and contained cells expressing neuronal and non‐neuronal proteins. To induce differentiation, precursor cells were cocultured with cochlear cells and/or cochlear supernatant. Differentiated precursor cells were immunopositive for specific hair cell markers, including myosin VIIa, FM1‐43, calretinin, phalloidin, and espin, and resembled hair cells anatomically and immunocytochemically in culture. The results demonstrate for the first time that adult olfactory precursor cells can differentiate into hair cell‐like cells, thus providing a potential autotransplantation therapy for hearing loss.

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.

Assignment of the human neuropeptide Y gene to chromosome 7p15.1 by nonisotopic in situ hybridization

A 15-kb genomic fragment containing the whole human NPY gene in the vector {lambda}GEM-11 was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 5 ng/{mu}l to metaphases from two males. The fluorescence in situ hybridization method was modified from that previously described in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhanced.

Galanin in human pituitary adenomas: frequency and clinical significance

objectives Galanin (GAL) is a neuropeptide widely expressed in the central and peripheral nervous system and in neuroendocrine tissue, including the adenohypophysis where, in humans, it is expressed in corticotrophs and in ACTH‐producing adenomas. Previous analyses of human tissue have used antiserum against porcine GAL for detection of GAL immunoreactivity (GAL‐IR) and no pathophysiological correlates have been reported. Given significant differences between the sequence of porcine and human GAL peptides, the aim of this study was to use antiserum raised against synthetic human GAL to investigate GAL‐IR in non tumorous pituitaries and in pituitary adenomas, and to correlate GAL‐IR with the clinical and hormonal characteristics of patients with Cushing’s disease.

Structural organization and chromosomal localization of three human galanin receptor genes.

Abstract: Human galanin receptor subtypes GALR1, GALR2, and GALR3 are encoded by separate genes that are located on human chromosomes 18q23, 17q25.3, and 22q13.1, respectively. The exon:intron organization of the gene encoding GALR2 (GALNR2) and GALR3 (GALNR3) is conserved, with exon 1 encoding the NH2‐terminus to the end of transmembrane domain 3 and exon 2 encoding the remainder of the receptor, from the second intracellular loop to the COOH‐terminus. This conservation of structural organization is indicative of a common evolutionary origin for GALNR2 and GALNR3. The exon:intron organization of the gene encoding GALR1 (GALNR1) is different from that of GALNR2 and GALNR3, with exon 1 encoding the NH2‐terminus to the end of transmembrane domain 5, exon 2 encoding the third intracellular loop, and exon 3 encoding the remainder of the receptor, from transmembrane domain 6 to the COOH‐terminus. The structural organization of GALNR1 suggests convergent evolution for this gene and represents a structural organization that is unique among genes encoding G‐protein‐coupled receptors.

Phenotypic analysis ofGalr1 knockout mice reveals a role for GALR1 galanin receptor in modulating seizure activity but not nerve regeneration

The GALR1 galanin receptor is expressed at high levels within the central nervous system and is hypothesised to play a significant role in many of the central actions of galanin. To determine which specific actions of galanin are mediated by GALR1, we have developed mice that carry an insertional inactivating mutation within the first coding exon of the gene encoding GALR1 (Galr1). HomozygousGalr1 −/− mice are viable. Both male and female mice exhibit reduced circulating levels of insulin-like growth factor-I (IGF-I) but no significant difference in growth rate relative toGalr1 +/+ controls. Female homozygousGalr1 −/− mice are capable of breeding and nursing offspring. Functional recovery after sciatic nerve crush is not significantly different inGalr1 −/− mice relative toGalr1 +/+ controls, indicating that GALR1 does not mediate the nerve regenerative effects of galanin. However, homozygousGalr1 −/− mice exhibit spontaneous seizures, identifying a critical role for GALR1 in mediating the anti-seizure activity of galanin.

Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder and is due to disease-causing variants in PKD1 or PKD2. Strong genotype–phenotype correlation exists although diagnostic sequencing is not part of routine clinical practice. This is because PKD1 bears 97.7% sequence similarity with six pseudogenes, requiring laborious and error-prone long-range PCR and Sanger sequencing to overcome. We hypothesised that whole-genome sequencing (WGS) would be able to overcome the problem of this sequence homology, because of 150 bp, paired-end reads and avoidance of capture bias that arises from targeted sequencing. We prospectively recruited a cohort of 28 unique pedigrees with ADPKD phenotype. Standard DNA extraction, library preparation and WGS were performed using Illumina HiSeq X and variants were classified following standard guidelines. Molecular diagnosis was made in 24 patients (86%), with 100% variant confirmation by current gold standard of long-range PCR and Sanger sequencing. We demonstrated unique alignment of sequencing reads over the pseudogene-homologous region. In addition to identifying function-affecting single-nucleotide variants and indels, we identified single- and multi-exon deletions affecting PKD1 and PKD2, which would have been challenging to identify using exome sequencing. We report the first use of WGS to diagnose ADPKD. This method overcomes pseudogene homology, provides uniform coverage, detects all variant types in a single test and is less labour-intensive than current techniques. This technique is translatable to a diagnostic setting, allows clinicians to make better-informed management decisions and has implications for other disease groups that are challenged by regions of confounding sequence homology.