Biomedical subjects

Robert E Hammer

Publications and source records attributed to Robert E Hammer.

At least 19 recordsLinked to original sources

PubMed2004

Double-knockout mice for alpha- and beta-synucleins: effect on synaptic functions.

An abundant presynaptic protein, alpha-synuclein, is centrally involved in the pathogenesis of Parkinson's disease. However, conflicting data exist about the normal function of alpha-synuclein, possibly because alpha-synuclein is redundant with the very similar beta-synuclein. To investigate the functions of synucleins systematically, we have now generated single- and double-knockout (KO) mice that lack alpha- and/or beta-synuclein. We find that deletion of synucleins in mice does not impair basic brain functions or survival. We detected no significant changes in the ultrastructure of synuclein-deficient synapses, in short- or long-term synaptic plasticity, or in the pool size or replenishment of recycling synaptic vesicles. However, protein quantitations revealed that KO of synucleins caused selective changes in two small synaptic signaling proteins, complexins and 14-3-3 proteins. Moreover, we found that dopamine levels in the brains of double-KO but not single-KO mice were decreased by approximately 20%. In contrast, serotonin levels were unchanged, and dopamine uptake and release from isolated nerve terminals were normal. These results show that synucleins are not essential components of the basic machinery for neurotransmitter release but may contribute to the long-term regulation and/or maintenance of presynaptic function.

Animals↗

PubMed2004

Defining the spermatogonial stem cell.

Through the use of donor cells from transgenic rats expressing GFP exclusively in the germline, we have defined culture conditions where male germ cells lose (on STO cells) or maintain (on MSC-1 cells) stem cell activity. A cadre of germ cell transcripts strikingly decrease in relative abundance as a function of testis age or culture time on STO cells, but only a subset of these transcripts (approximately 248) remain elevated when cultured on MSC-1 cells. If specific gene expression regulates stem cell activity, some or all of these transcripts are candidates as such regulators. We establish a spermatogonial stem cell index (SSCI) that reliably predicts relative stem cell activity in rat or mouse testis cell cultures, and through the use of an antibody to a robust signal (Egr3) within the index find intense signals in single or paired cells. As germ cells form longer interconnected chains (incomplete cytokinesis), the Egr3 signal disappears coincident with a loss of stem cell activity. Thus, molecular markers specific for spermatogonial stem cells establish a reliable and rapid means by which to define these cells in culture and alleviate the need for laborious testicular transfers in initial cell culture studies.

Animals↗

PubMed2004

Expression of a poly-glutamine-ataxin-3 transgene in orexin neurons induces narcolepsy-cataplexy in the rat.

The sleep disorder narcolepsy has been linked to loss of hypothalamic neurons producing the orexin (hypocretin) neuropeptides. Here, we report the generation of transgenic rats expressing a human ataxin-3 fragment with an elongated polyglutamyl stretch under control of the human prepro-orexin promoter (orexin/ataxin-3 rats). At 17 weeks of age, the transgenic rats exhibited postnatal loss of orexin-positive neurons in the lateral hypothalamus, and orexin-containing projections were essentially undetectable. The loss of orexin production resulted in the expression of a phenotype with fragmented vigilance states, a decreased latency to rapid eye movement (REM) sleep and increased REM sleep time during the dark active phase. Wakefulness time was also reduced during the dark phase, and this effect was concentrated at the photoperiod boundaries. Direct transitions from wakefulness to REM sleep, a defining characteristic of narcolepsy, occurred frequently. Brief episodes of muscle atonia and postural collapse resembling cataplexy were also noted while rats maintained the electroencephalographic characteristics of wakefulness. These findings indicate that the orexin/ataxin-3 transgenic rat could provide a useful model of human narcolepsy.

Animals↗

PubMed2004

Disregulated glyceroneogenesis: PCK1 as a candidate diabetes and obesity gene.

Genetics and diet interact to cause type 2 diabetes mellitus and obesity. PCK1 has been implicated as one of many genes associated with type 2 diabetes mellitus. The common assumption is that mutations in PCK1 lead to excessive glucose production through hepatic gluconeogenesis. However, there is an alternative explanation, wherein mutations at the PCK1 locus could selectively affect PCK1 expression in adipose tissue. The result would be changes in glyceroneogenesis that would affect the storage and release of fatty acids. Here, we present the novel hypothesis that a variety of phenotypes could arise from mutations of the various tissue-specific control elements of PCK1. We also suggest specific quantitative metabolic traits that would accompany mutations that selectively affect PCK1 expression in adipose tissue.

Diabetes Mellitus↗

PubMed2004

Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1.

LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis.

In the current studies we generated transgenic mice that overexpress human Insig-1 in the liver under a constitutive promoter. In cultured cells Insig-1 and Insig-2 have been shown to block lipid synthesis in a cholesterol-dependent fashion by inhibiting proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate lipid synthesis. Insig's exert this action in the ER by binding SREBP cleavage-activating protein (SCAP) and preventing it from escorting SREBPs to the Golgi apparatus where the SREBPs are processed to their active forms. In the livers of Insig-1 transgenic mice, the content of all nuclear SREBPs (nSREBPs) was reduced and declined further upon feeding of dietary cholesterol. The nuclear content of the insulin-induced SREBP isoform, SREBP-1c, failed to increase to a normal extent upon refeeding on a high-carbohydrate diet. The nSREBP deficiency produced a marked reduction in the levels of mRNAs encoding enzymes required for synthesis of cholesterol, fatty acids, and triglycerides. Plasma cholesterol levels were strongly reduced, and plasma triglycerides did not exhibit their normal rise after refeeding. These results provide in vivo support for the hypothesis that nSREBPs are essential for high levels of lipid synthesis in the liver and indicate that Insig's modulate nSREBP levels by binding and retaining SCAP in the ER.

Animals↗

PubMed2003

Hyperactivated sperm motility driven by CatSper2 is required for fertilization.

Elevations of sperm Ca2+ seem to be responsible for an asymmetric form of motility called hyperactivation, which is first seen near the time of fertilization. The mechanism by which intracellular Ca2+ concentrations increase remains unknown despite considerable investigation. Although several prototypical voltage-gated calcium channels are present in spermatozoa, they are not essential for motility. Furthermore, the forward velocity and percentage of motility of spermatozoa are associated with infertility, but their importance relative to hyperactivation also remains unknown. We show here that disruption of the gene for a recently described sperm-specific voltage-gated cation channel, CatSper2, fails to significantly alter sperm production, protein tyrosine phosphorylation that is associated with capacitation, induction of the acrosome reaction, forward velocity, or percentage of motility, yet CatSper2-/- males are completely infertile. The defect that we identify in the null sperm cells is a failure to acquire hyperactivated motility, which seems to render spermatozoa incapable of generating the "power" needed for penetration of the extracellular matrix of the egg. A loss of power is suggested also by experiments in which the viscosity of the medium was increased after incubation of spermatozoa in normal capacitating conditions. In high-viscosity medium, CatSper2-null spermatozoa lost the ability to swim forward, whereas wild-type cells continued to move forward. Thus, CatSper2 is responsible for driving hyperactivated motility, and, even with typical sperm forward velocities, fertilization is not possible in the absence of this highly active form of motility.

Animals↗

PubMed2003

Cell-autonomous and nonautonomous actions of endothelin-A receptor signaling in craniofacial and cardiovascular development.

Craniofacial and cardiac development relies on the proper patterning of the neural crest-derived ectomesenchyme of the pharyngeal arches, from which many craniofacial and great vessel structures arise. One of the intercellular signaling molecules that is involved in this process, endothelin-1 (ET-1), is expressed in the arch epithelium and influences arch development by binding to its cognate receptor, the endothelin A (ET(A)) receptor, found on ectomesenchymal cells. We have previously shown that absence of ET(A) signaling in ET(A)(-/-) mouse embryos disrupts neural crest cell development, resulting in craniofacial and cardiovascular defects similar in many aspects to those in mouse models of DiGeorge syndrome. These changes may reflect a cell-autonomous requirement for ET(A) signaling during crest cell development because the ET(A) receptor is an intracellular signaling molecule. However, it is also possible that some of the observed defects in ET(A)(-/-) embryos could arise from the absence of downstream signaling that act in a non-cell-autonomous manner. To address this question, we performed chimera analysis using ET(A)(-/-) embryonic stem cells. We observe that, in almost all early ET(A)(-/-) --> (+/+) chimeric embryos, ET(A)(-/-) cells are excluded from the caudoventral aspects of the pharyngeal arches, suggesting a cell-autonomous role for ET(A) signaling in crest cell migration and/or colonization. Interestingly, in the few embryos in which mutant cells do reach the ventral arch, structures derived from this area are either composed solely of wild type cells or are missing, suggesting a second cell-autonomous role for ET(A) signaling in postmigratory crest cell differentiation. In the cardiac outflow tract and great vessels, ET(A)(-/-) cells are excluded from the walls of the developing pharyngeal arch arteries, indicating that ET(A) signaling also acts cell-autonomously during cardiac neural crest cell development.

Animals↗

PubMed2003

Overexpression of sterol regulatory element-binding protein-1a in mouse adipose tissue produces adipocyte hypertrophy, increased fatty acid secretion, and fatty liver.

Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate cholesterol and fatty acid homeostasis. In mammals, three SREBP isoforms designated SREBP-1a, SREBP-1c, and SREBP-2 have been identified. SREBP-1a and SREBP-1c are derived from the same gene by virtue of alternatively spliced first exons. SREBP-1a has a longer transcriptional activation domain and is a more potent transcriptional activator than SREBP-1c in cultured cells and liver. Here, we describe the physiologic consequences of overexpressing the nuclear form of SREBP-1a (nSREBP-1a) in adipocytes of mice using the adipocyte-specific aP2 promoter (aP2-nSREBP-1a). The transgenic aP2-nSREBP-1a mice developed markedly enlarged white and brown adipocytes that were fully differentiated. Adipocytes isolated from aP2-nSREBP-1a mice had significantly increased rates of fatty acid synthesis and enhanced fatty acid secretion. The increased production and release of fatty acids from adipocytes led, in turn, to a fatty liver. Overexpression of the alternative SREBP-1 isoform, nSREBP-1c, in adipose tissue inhibits adipocyte differentiation; as a result, the transgenic nSREBP-1c mice develop a syndrome resembling human lipodystrophy, which includes a loss of peripheral white adipose tissue, diabetes, and fatty livers (Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y., Goldstein, J. L., and Brown, M. S. (1998) Genes Dev. 12, 3182-3194). In striking contrast, nSREBP-1a overexpression in fat resulted in the hypertrophy of fully differentiated adipocytes, no diabetes, and mild hepatic steatosis. These results suggest that nSREBP-1a and nSREBP-1c have distinct roles in adipocyte fat metabolism in vivo.

Adipocytes↗

PubMed2003

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis.

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.

Animals↗

PubMed2003

Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes.

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.

Animals↗

PubMed2003

dHAND-Cre transgenic mice reveal specific potential functions of dHAND during craniofacial development.

Most of the bone, cartilage, and connective tissue of the craniofacial region arise from cephalic neural crest cells. Presumably, patterning differences in crest cells are a result of regional action of transcription factors within the developing pharyngeal arches. The basic helix-loop-helix transcription factor dHAND/HAND2 is expressed throughout much of the neural crest-derived mesenchyme of the pharyngeal arches, suggesting that it plays a crucial role in craniofacial development. However, targeted inactivation of the dHAND gene results in embryonic lethality by E10.5 due to vascular defects, preventing further analysis of the role of dHAND in cephalic neural crest cell development. In order to examine putative roles of dHAND during later stages of embryogenesis, we have used a transgenic lineage marker approach, in which a portion of the dHAND upstream region containing an enhancer that directs dHAND expression to the pharyngeal arches is used to drive Cre recombinase expression. By crossing these dHAND-Cre transgenic mice with R26R mice, we can follow the fate of cells that expressed dHAND at any time during development by examining beta-galactosidase activity. We show that dHAND is first expressed in postmigratory cephalic neural crest cells within the pharyngeal arches. In older embryos, beta-galactosidase-labeled cells are observed in most of the neural crest-derived lower jaw skeleton and surrounding connective tissues. However, labeled cells only contribute to substructures within the middle ear, indicating that our transgene is not globally expressed in cephalic neural crest cells within the pharyngeal arches. Moreover, dHAND-Cre mice will provide a valuable tool for tissue-specific inactivation of gene expression in multiple tissue types of neural crest origin.

Animals↗

PubMed2003

Normal sorting but defective endocytosis of the low density lipoprotein receptor in mice with autosomal recessive hypercholesterolemia.

Autosomal recessive hypercholesterolemia (ARH) is a genetic form of hypercholesterolemia that clinically resembles familial hypercholesterolemia (FH). As in FH, the rate of clearance of circulating low density lipoprotein (LDL) by the LDL receptor (LDLR) in the liver is markedly reduced in ARH. Unlike FH, LDL uptake in cultured fibroblasts from ARH patients is normal or only slightly impaired. The gene defective in ARH encodes a putative adaptor protein that has been implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in the liver, ARH-deficient mice were developed. Plasma levels of LDL-cholesterol were elevated in the chow-fed Arh-/- mice (83 +/- 8 mg/dl versus 68 +/- 8 mg/dl) but were lower than those of mice expressing no LDLR (Ldlr-/-) (197 +/- 8 mg/dl). Cholesterol feeding elevated plasma cholesterol levels in both strains. The fractional clearance rate of radiolabeled LDL was reduced to similar levels in the Arh-/- and Ldlr-/- mice, whereas the rate of removal of alpha2-macroglobulin by the LDLR-related protein, which also interacts with ARH, was unchanged. Immunolocalization studies revealed that a much greater proportion of immunodetectable LDLR, but not LDLR-related protein, was present on the sinusoidal surface of hepatocytes in the Arh-/- mice. Taken together, these results are consistent with ARH playing a critical and specific role in LDLR endocytosis in the liver.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Induction of hepatocyte proliferation and death by modulation of T-Antigen expression.

Mice expressing SV40 T-Antigen in liver under control of the phosphoenolpyruvate carboxykinase promoter were generated. By altering the carbohydrate content of the diet, TAg expression, the rate of hepatocyte proliferation and apoptosis, and hence hepatocarcinogenesis, could be regulated. Carbohydrate-mediated suppression of TAg resulted in slow hepatic growth that progressed to focal hepatocellular carcinoma (HCC) after a long latency period. In contrast, induction of TAg by feeding mice a low carbohydrate diet resulted in massive hepatomegaly that progressed rapidly to diffuse multifocal HCC. Hepatic TAg expression could be efficiently repressed by switching mice from the low to the high-carbohydrate diet, which if instigated prior to the development of HCC, resulted in rapid regression through a p53-independent reduction in hepatocyte proliferation and an increase in hepatocyte apoptosis. Although liver growth was accompanied by compensatory hepatocyte apoptosis, an apoptotic deficit developed following chronic exposure to high levels of TAg. This was associated with Akt phosphorylation and increased expression of the antiapoptotic molecules bfl-1/A1, TIAP, and A20. Mice were resistant to Fas-induced hepatocellular apoptosis due to severely impaired caspase activation and failed activation of the mitochondrial amplification loop. This model will be useful to investigate oncogene-mediated disruption of the cell cycle and apoptosis, and to determine which processes constitute fixed, or reversible aspects of the tumorigenic process.

Animals↗

PubMed2003

A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission.

Mints (also called X11-like proteins) are adaptor proteins composed of divergent N-terminal sequences that bind to synaptic proteins such as CASK (Mint 1 only) and Munc18-1 (Mints 1 and 2) and conserved C-terminal PTB- and PDZ-domains that bind to widely distributed proteins such as APP, presenilins, and Ca(2+) channels (all Mints). We find that Mints 1 and 2 are similarly expressed in most neurons except for inhibitory interneurons that contain selectively high levels of Mint 1. Using knockout mice, we show that deletion of Mint 1 does not impair survival or alter the overall brain architecture, arguing against an essential developmental function of the Mint 1-CASK complex. In electrophysiological recordings in the hippocampus, we observed no changes in short- or long-term synaptic plasticity in excitatory synapses from Mint 1-deficient mice and detected no alterations in the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to N-methyl-d-aspartate (NMDA) receptor-mediated synaptic currents. Thus the Mint 1-CASK complex is not required for AMPA- and NMDA-receptor functions or for synaptic plasticity in excitatory synapses. In inhibitory synapses, however, we uncovered an approximately 3-fold increase in presynaptic paired-pulse depression, suggesting that deletion of Mint 1 impairs the regulation of gamma-aminobutyric acid release. Our data indicate that Mints 1 and 2 perform redundant synaptic functions that become apparent in Mint 1-deficient mice in inhibitory interneurons because these neurons selectively express higher levels of Mint 1 than Mint 2.

Adaptor Proteins, Signal Transducing↗

PubMed2003

RNA amplification and transcriptional profiling for analysis of stem cell populations.

As stem cells constitute a rare cell pool, a global analysis of gene expression is typically not possible. Here, we use a T7-based RNA amplification method combined with high-density oligonucleotide array technology to analyze gene expression. We isolated RNA from ES cells (100-100,000 cells), subjected them to two rounds of amplification, and compared each sample using microarray analysis. RNA isolation and amplification was highly reproducible and sensitive for the analysis of low abundant transcripts. Greater than 93% of the transcripts expressed were changed less than 2-fold when comparing the results of amplified RNA (100 cells to 100,000 cells) to unamplified RNA (isolated from 1,000,000 cells). This transcriptional analysis resulted in minimal skewing of gene expression. Using this technology, we analyzed the genetic programs of ES cells, STO feeder cells, and adult cardiomyocytes. We conclude that these technologies can be applied to the analysis of the genetic programs of rare cell populations and ultimately this analysis will enhance our understanding of the regulatory mechanisms of stem cell populations.

Animals↗

PubMed2003

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain.

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.

Animals↗

PubMed2003

Regulation of cytosolic phosphoenolpyruvate carboxykinase gene expression in adipocytes.

Cytosolic phosphoenolpyruvate carboxykinase (EC 4.1.1.32; PEPCK-C) catalyzes the critical regulated step in adipocyte glyceroneogenesis. Numerous studies have shown that hormones and nutrients regulate PEPCK-C at the transcriptional level. We identified two upstream cis-acting DNA elements, gAF1/PCK1 and PCK2, that control adipocyte specific transcription of the PEPCK-C gene (Pck1). Both elements are direct repeat hexanucleotides separated by 1 bp (DR1 elements; variations of the sequence AGGTCAnAGGTCA). PCK2 is located 1 kbp upstream and is the essential element of an adipocyte specific enhancer. It is a peroxisome proliferator activated receptor gamma response element (PPRE) and directs the activation of the PEPCK-C gene during adipogenesis. In addition, it is a thiazolidinedione response element in mature adipocytes. In contrast, gAF1/PCK1, centered 445 bp upstream, is a pleiotropic element that mediates tissue specific glucocorticoid action-repression in adipocytes and induction in hepatocytes. It is a negative response element for PPARgamma, RXRalpha, COUP-TFII, and several unidentified proteins in some cell types, and a positive element for COUP-TFI and HNF4 in other cells type. The purpose of this presentation is to review the discovery and characterization of these two elements in adipocytes and describe how our work has contributed to understanding the mechanisms that control adipocyte glyceroneogenesis.

Adipocytes↗