Neuroanatomical and functional characterization of CRF neurons of the amygdala using a novel transgenic mouse model.

The corticotropin-releasing factor (CRF)-producing neurons of the amygdala have been implicated in behavioral and physiological responses associated with fear, anxiety, stress, food intake and reward. To overcome the difficulties in identifying CRF neurons within the amygdala, a novel transgenic mouse line, in which the humanized recombinant Renilla reniformis green fluorescent protein (hrGFP) is under the control of the CRF promoter (CRF-hrGFP mice), was developed. First, the CRF-hrGFP mouse model was validated and the localization of CRF neurons within the amygdala was systematically mapped. Amygdalar hrGFP-expressing neurons were located primarily in the interstitial nucleus of the posterior limb of the anterior commissure, but also present in the central amygdala. Secondly, the marker of neuronal activation c-Fos was used to explore the response of amygdalar CRF neurons in CRF-hrGFP mice under different experimental paradigms. C-Fos induction was observed in CRF neurons of CRF-hrGFP mice exposed to an acute social defeat stress event, a fasting/refeeding paradigm or lipopolysaccharide (LPS) administration. In contrast, no c-Fos induction was detected in CRF neurons of CRF-hrGFP mice exposed to restraint stress, forced swimming test, 48-h fasting, acute high-fat diet (HFD) consumption, intermittent HFD consumption, ad libitum HFD consumption, HFD withdrawal, conditioned HFD aversion, ghrelin administration or melanocortin 4 receptor agonist administration. Thus, this study fully characterizes the distribution of amygdala CRF neurons in mice and suggests that they are involved in some, but not all, stress or food intake-related behaviors recruiting the amygdala.

The P7C3 class of neuroprotective compounds exerts antidepressant efficacy in mice by increasing hippocampal neurogenesis.

Augmenting hippocampal neurogenesis represents a potential new strategy for treating depression. Here we test this possibility by comparing hippocampal neurogenesis in depression-prone ghrelin receptor (Ghsr)-null mice to that in wild-type littermates and by determining the antidepressant efficacy of the P7C3 class of neuroprotective compounds. Exposure of Ghsr-null mice to chronic social defeat stress (CSDS) elicits more severe depressive-like behavior than in CSDS-exposed wild-type littermates, and exposure of Ghsr-null mice to 60% caloric restriction fails to elicit antidepressant-like behavior. CSDS resulted in more severely reduced cell proliferation and survival in the ventral dentate gyrus (DG) subgranular zone of Ghsr-null mice than in that of wild-type littermates. Also, caloric restriction increased apoptosis of DG subgranular zone cells in Ghsr-null mice, although it had the opposite effect in wild-type littermates. Systemic treatment with P7C3 during CSDS increased survival of proliferating DG cells, which ultimately developed into mature (NeuN+) neurons. Notably, P7C3 exerted a potent antidepressant-like effect in Ghsr-null mice exposed to either CSDS or caloric restriction, while the more highly active analog P7C3-A20 also exerted an antidepressant-like effect in wild-type littermates. Focal ablation of hippocampal stem cells with radiation eliminated this antidepressant effect, further attributing the P7C3 class antidepressant effect to its neuroprotective properties and resultant augmentation of hippocampal neurogenesis. Finally, P7C3-A20 demonstrated greater proneurogenic efficacy than a wide spectrum of currently marketed antidepressant drugs. Taken together, our data confirm the role of aberrant hippocampal neurogenesis in the etiology of depression and suggest that the neuroprotective P7C3-compounds represent a novel strategy for treating patients with this disease.

Characterization of Kiss1 neurons using transgenic mouse models.

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or ß-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Infrequency of BRCA2 alterations in head and neck squamous cell carcinoma.

Alterations of BRCA2 result in increased susceptibility to breast cancer in both men and women (relative lifetime risks of 0.06 and 0.8 respectively). BRCA2 maps to 13q12-q13 and encodes a transcript of 10,157 bp. Other cancers that have been described in BRCA2 mutation carriers include those of the larynx. Human chromosome 13q has been shown previously by LOH studies to harbor several tumor suppressor genes for head and neck squamous cell carcinoma (HNSCCs). We therefore examined the role of BRCA2 in the development of these cancers. Only 6/22 (27%) of the laryngeal cancers we examined demonstrated LOH of the BRCA2-containing region. These and 10 other HNSCCs of different origins that were demonstrated by LOH studies to have lost the region of chromosome 13 containing BRCA2 were examined for alterations in this gene. SSCP analysis failed to reveal any alterations leading us to conclude that BRCA2 alterations are not frequently involved in the pathogenesis of HNSCCs and that the observed LOH of chromosome 13 loci is due to other, as yet, unidentified tumor suppressor gene(s). Interestingly tumors with LOH in this region (proximal to D13S118) were far more likely to be derived from women than men. This is unusual since HNSCCs are usually fourfold more common in men than in women.

Characterization of EZH1, a human homolog of Drosophila Enhancer of zeste near BRCA1.

Recent transcription mapping efforts within chromosome 17q21 have led to the identification of a human homolog of the Drosophila gene Enhancer of zeste, E(z). A member of the Polycomb group (Pc-G) of proteins, Drosophila E(z) acts as a negative regulator of the segment identity genes of the Antennapedia and Bithorax complexes. Here we report the full-length protein coding sequence of human EZH1 (Enhancer of zeste homolog 1) and compare the respective protein sequences in both species. EZH1 encodes a protein of 747 amino acids that displays 55% amino acid identity overall (70% similarity) with Drosophila E(z). The strongest homology was noted (79% identity, 89% similarity) within the carboxy-terminal 245 amino acids, including the SET domain, a region of E(z) also conserved in other Drosophila proteins with roles in development and/or chromatin structure. A large Cysrich region with a novel spatial pattern of cysteine residues was also conserved in both EZH1 and E(z). The strong sequence conservation suggest potential roles for EZH1 in human development as a transcriptional regulator and as a component of protein complexes that stably maintain heterochromatin. EZH1 is expressed as two major transcripts in all adult and fetal human tissues surveyed; comparison of cloned cDNAs suggests that alternative splicing may account for at least part of the transcript size difference. Analysis of one cDNA revealed an unusual splicing event involving EZH1 and a tandemly linked gene GPR2 and suggests a potential mechanism for modifying the EZH1 protein in the conserved C-terminal domain. The sequence and isolated cDNAs will provide useful reagents for determining the function of EZH1 and the importance of the evolutionarily conserved domains.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Direct selection of expressed sequences within a 1-Mb region flanking BRCA1 on human chromosome 17q21.

Direct selection of genes within the interval of chromosome 17q21 containing BRCA1 was performed. YAC and cosmid contigs spanning the BRCA1 region were used to select cDNA clones from pools of cDNAs derived from human placenta, HeLa cells, activated T cells, and fetal head. A minimum set of 48 fragments of nonoverlapping cDNAs that unequivocally mapped within a 1-Mb region was identified, although it is not yet known how many of these are derived from the same transcript. DNA sequence analyses revealed that 4 of these cDNAs were derived from known genes (EDH17B2, glucose-6-phosphatase, IAI.3B, and E1AF), 1 is a member of a previously described gene family (HMG-17), and 7 share substantial identity with previously described genes from human or other species. The remainder showed no significant homology to known genes. Limited PCR-based expression profiles of a set of 13 of the genes were performed, and all gave positive results with at least some cDNA sources supporting the contention that they truly represent transcribed sequences. A comparison between genes obtained from this region by direct selection with those obtained by direct screening or exon trapping (see accompanying papers, this issue) revealed that over 90% of the genes identified by exon trapping were represented in the selected material and that at least two additional genes that appear to represent low abundance transcripts with restricted expression profiles were identified by selection but not by other means.

A somatic cell hybrid map of human chromosome 13.

We have constructed a chromosome 13 somatic cell hybrid map using seven cell lines: PGMEA6, a hybrid containing the entire chromosome 13, and six hybrids containing various deletions of chromosome 13 (BARF7, PPF22, KBF11, KSF39, CF25, and CF27). We have mapped 80 markers that define 10 regions of chromosome 13 with respect to 10 breakpoints in the mapping panel; these regions range in size from 4 to 24 Mb, with an average size of 8 Mb. The 80 markers sublocalized on our mapping panel include 10 Alu-PCR clones, 6 of which were converted to sequence-tagged sites; 40 (CA)n repeat-containing clones, 27 of which are microsatellite PCR markers; 8 (AAAG)n repeat-containing PCR markers, 1 two-allele PCR marker, 4 genes or expressed sequences, and 17 anonymous DNA probes. This low-resolution physical map can be used as a backbone map for more refined physical mapping using radiation hybrids or yeast artificial chromosomes.

Polymorphic microsatellites and Wilson disease (WD).

Wilson disease (WD), an autosomal recessive disorder of copper metabolism, has been previously mapped to chromosome 13q. Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) at 13q14.3, as well as sequence-tagged sites for closely linked loci, are described. Two polymorphic microsatellite markers at D13S118 and D13S119 lie within 3 cM of WND. Two others (D13S227 and D13S228) were derived from a yeast artificial chromosome containing D13S31. These were placed on a genetic linkage map of chromosome 13 and were typed in 74 multiplex WD families from a variety of geographic origins (166 affected members). Multipoint analysis provides very high odds that the location of WND is between D13S31/D13S227/D13S228 and D13S59. Previous odds with RFLP-based markers were only 7:1 more likely than any other location. Current odds are 5,000:1. Preclinical testing of three cases of WD by using the highly informative polymorphic microsatellite markers is described. The markers described here ensure that 95% of predictive tests using DNA from both parents and from at least one affected sib will have an accuracy > 99%.

High-density genetic map of the BRCA1 region of chromosome 17q12-q21.

To facilitate the positional cloning of the breast-ovarian cancer gene BRCA1, we constructed a high-density genetic map of the 8.3-cM interval between D17S250 and GIP on chromosome 17q12-q21. Markers were mapped by linkage in the CEPH and in extended kindreds in our breast cancer series. The map comprises 33 ordered polymorphisms, including 12 genes and 21 anonymous markers, yielding an average of one polymorphism every 250 kb. Twenty-five of the markers are PCR-based systems. The order of polymorphic genes and markers is cen-D17S250-D17S518-HER2-THRA1-RARA-D17S80 -KRT10-[D17S800-D17S857]-GAS- D17S856-EDH17B-D17S855-D17S859-D17S858-[++ +PPY-D17S78]-D17S183-EPB3-D17S579- D17S509-[D17S508-D17S190 = D17S810]-D17S791-[D17S181 = D17S806]-D17S797- HOX2B-GP3A-[D17S507 = GIP]-qter. BRCA1 lies in the middle of the interval, between THRA1 and D17S183. Markers from this map can be used to determine whether cancer is linked to BRCA1 in families, to evaluate whether tumors have lost heterozygosity at loci in the region, and to identify probes for characterizing chromosomal rearrangements from patients and from tumors.

THRA1 and D17S183 flank an interval of < 4 cM for the breast-ovarian cancer gene (BRCA1) on chromosome 17q21.

In order to pinpoint the locale of the gene for early-onset familial breast and ovarian cancer (BRCA1), polymorphisms were developed within the locus for thyroid hormone receptor alpha (THRA1) and for several anonymous sequences at chromosome 17q12-q21. The THRA1 polymorphism is a dinucleotide repeat with 10 alleles and heterozygosity.79. Gene mapping in extended families with inherited, early-onset breast and ovarian cancer indicates that BRCA1 is distal to THRA1 and proximal to D17S183 (SCG43), an interval of < 4 cM. This locale excludes HER2, THRA1, WNT3, HOX2, NGFR, PHB, COLIA1, NME1, and NME2 as candidates for BRCA1 but does not exclude RARA or EDH17B. Resolving the remaining recombination events in these families by new polymorphisms in the THRA1-D17S183 interval will facilitate positional cloning of the breast-ovarian cancer gene on chromosome 17q12-q21.

Microsatellite polymorphism linkage map of human chromosome 13q.

Twelve polymorphic (CA)n microsatellites were isolated from a flow-sorted chromosome 13 genomic library. These, and two others that have been previously described, were genotyped in 41 families from the CEPH (Centre d'Etude Polymorphisme Humain, Paris), and a primary linkage map with considerable support for order (odds > 10,000:1) was constructed. Two RFLP-based markers, COL4A1 and D13S52, with heterozygosities above 0.67 and an RFLP-based centromeric marker at D13Z1 were included in this map which extends from 13cen to 13q34. The heterozygosity of all of the PCR-based markers is above 60%. The total map spans a genetic distance of 144 cM, extending from D13Z1 to D13S52 with a single maximum intermarker recombination distance of 35 cM. All other intermarker recombination distances are 18 cM or less. Marker order was confirmed by sublocalizing many of the microsatellite containing clones on a panel of rodent-human somatic cell hybrids with deletions and rearrangements of chromosome 13. One spontaneous new mutation for these 14 (CA)n repeat markers was identified from a total of 8006 gametes, giving an overall observed spontaneous mutation rate of 0.00012 per locus per gamete. An integrated map of chromosome 13q was constructed with the microsatellite markers described here and previously genotyped RFLP-based markers. This sex-average map spans 209 cM with an average distance between unique map locations of 4.5 cM; the maximum intermarker distance was 14 cM.

Structure and chromosomal location of the human gene encoding cartilage matrix protein.

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene.

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.

Clonal heterogeneity of synovial fluid T lymphocytes from patients with rheumatoid arthritis.

Although substantial evidence suggests that synovial T lymphocytes are critical in the pathogenesis of rheumatoid arthritis (RA), little is known regarding their antigenic specificities, antigen receptor gene rearrangements, and mechanisms of activation. To assess the extent of expansion of specific clones among RA synovial fluid T cells, Southern blot analyses of T-cell receptor (TCR) gene rearrangements were performed on 40 RA synovial fluid T-cell clones, as well as on both fresh and polyclonally activated T cells from RA synovial fluid, RA peripheral blood, and normal peripheral blood. Two of the clones had identical TCR rearrangement patterns, but the remainder were unique. The nonclonal RA T-cell samples showed the same pattern of TCR beta-chain rearrangement that was observed among normal peripheral blood T cells, indicating no dominant clonal T-cell population in these samples. It was noted that with sufficient exposure of autoradiograms of the Southern blots, discrete TCR gene rearrangements, representing in some cases common D beta J beta (D, diversity; J, joining) rearrangements, were evident in T cells from peripheral blood of normal individuals and patients with RA, as well as T cells from RA synovial fluid. Taken together, the findings indicate that only a minor degree of oligoclonality can be demonstrated among T lymphocytes from RA synovial fluid.