Carrolee Barlow

Atm-Deficient Mice: A Paradigm of Ataxia Telangiectasia

A murine model of ataxia telangiectasia was created by disrupting the Atm locus via gene targeting. Mice homozygous for the disrupted Atm allele displayed growth retardation, neurologic dysfunction, male and female infertility secondary to the absence of mature gametes, defects in T lymphocyte maturation, and extreme sensitivity to gamma-irradiation. The majority of animals developed malignant thymic lymphomas between 2 and 4 months of age. Several chromosomal anomalies were detected in one of these tumors. Fibroblasts from these mice grew slowly and exhibited abnormal radiation-induced G1 checkpoint function. Atm-disrupted mice recapitulate the ataxia telangiectasia phenotype in humans, providing a mammalian model in which to study the pathophysiology of this pleiotropic disorder.

Elevated gene expression levels distinguish human from non-human primate brains

Little is known about how the human brain differs from that of our closest relatives. To investigate the genetic basis of human specializations in brain organization and cognition, we compared gene expression profiles for the cerebral cortex of humans, chimpanzees, and rhesus macaques by using several independent techniques. We identified 169 genes that exhibited expression differences between human and chimpanzee cortex, and 91 were ascribed to the human lineage by using macaques as an outgroup. Surprisingly, most differences between the brains of humans and non-human primates involved up-regulation, with ≈90% of the genes being more highly expressed in humans. By contrast, in the comparison of human and chimpanzee heart and liver, the numbers of up- and down-regulated genes were nearly identical. Our results indicate that the human brain displays a distinctive pattern of gene expression relative to non-human primates, with higher expression levels for many genes belonging to a wide variety of functional classes. The increased expression of these genes could provide the basis for extensive modifications of cerebral physiology and function in humans and suggests that the human brain is characterized by elevated levels of neuronal activity.

Increased Hepatic Cell Proliferation and Lung Abnormalities in Mice Deficient in CCAAT/Enhancer Binding Protein α

CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor that has been implicated in the regulation of cell-specific gene expression mainly in hepatocytes and adipocytes but also in several other terminally differentiated cells. It has been previously demonstrated that the C/EBPα protein is functionally indispensable, as inactivation of the C/EBPα gene by homologous recombination in mice results in the death of animals homozygous for the mutation shortly after birth (Wang, N., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112). Here we show that C/EBPα −1−mice have defects in the control of hepatic growth and lung development. The liver architecture is disturbed, with acinar formation, in a pattern suggestive of either regenerating liver or pseudoglandular hepatocellular carcinoma. Pulmonary histology shows hyperproliferation of type II pneumocytes and disturbed alveolar architecture. At the molecular level, accumulation of glycogen and lipids in the liver and adipose tissues is impaired, and the mutant animals are severely hypoglycemic. Levels of c-myc and c-jun RNA are specifically induced by several fold in the livers of the C/EBPα −/− animals, indicating an active proliferative stage. Furthermore, immunohistologic detection with an antibody to proliferating cell nuclear antigen/cyclin shows a 5-10 times higher frequency of positively stained hepatocytes in C/EBPα −/− liver. These results suggest a critical role for C/EBPα in vivo for the acquisition of terminally differentiated functions in liver including the maintenance of physiologic energy homeostasis.

Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1α Reduces Rather Than Increases Hypoxic-Ischemic Damage

Hypoxia-inducible factor-1α (HIF-1α) plays an essential role in cellular and systemic O2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1α in the brain, we exposed adult mice with late-stage brain deletion of HIF-1α to hypoxic injuries. Contrary to expectations, the brains from the HIF-1α-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1α can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1α-deficient mice. Although the role of HIF-1α has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1α in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1α-deficient mice is mechanistically consistent with a predominant role of HIF-1α as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1α, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

A targeted dominant negative mutation of the thyroid hormone α1 receptor causes increased mortality, infertility, and dwarfism in mice

Mutations in the thyroid hormone receptor β (TRβ) gene result in resistance to thyroid hormone. However, it is unknown whether mutations in the TRα gene could lead to a similar disease. To address this question, we prepared mutant mice by targeting mutant thyroid hormone receptor kindred PV (PV) mutation to the TRα gene locus by means of homologous recombination (TRα1PV mice). The PV mutation was derived from a patient with severe resistance to thyroid hormone that has a frameshift of the C-terminal 14 aa of TRβ1. We knocked in the same PV mutation to the corresponding TRα gene locus to compare the phenotypes of TRα1PV/+ mice with those of TRβPV/+ mice. TRα1PV/+ mice were viable, indicating that the mutation of the TRα gene is not embryonic lethal. In drastic contrast to the TRβPV/+ mice, which do not exhibit a growth abnormality, TRα1PV/+ mice were dwarfs. These dwarfs exhibited increased mortality and reduced fertility. In contrast to TRβPV/+ mice, which have a hyperactive thyroid, TRα1PV/+ mice exhibited mild thyroid failure. The in vivo pattern of abnormal regulation of T3 target genes in TRα1PV/+ mice was unique from those of TRβPV/+ mice. The distinct phenotypes exhibited by TRα1PV/+ and TRβPV/+ mice indicate that the in vivo functions of TR mutants are isoform-dependent. The TRα1PV/+ mice may be used as a tool to uncover human diseases associated with mutations in the TRα gene and, furthermore, to understand the molecular mechanisms by which TR isoforms exert their biological activities.

Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell–dependent antigen (Ag), Atm −/− mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm +/+ controls. To determine whether Atm −/− B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm −/− cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm −/− B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm −/− B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm −/− B cells occurs at the level of genomic DNA recombination as measured by digestion–circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the μ–γ1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity

Neuropathy target esterase (NTE) is involved in neural development and is the target for neurodegeneration induced by selected organophosphorus pesticides and chemical warfare agents. We generated mice with disruptions in Nte, the gene encoding NTE. Nte−/− mice die after embryonic day 8, and Nte+/− mice have lower activity of Nte in the brain and higher mortality when exposed to the Nte-inhibiting compound ethyl octylphosphonofluoridate (EOPF) than do wild-type mice. Nte+/− and wild-type mice treated with 1 mg per kg of body weight of EOPF have elevated motor activity, showing that even minor reduction of Nte activity leads to hyperactivity. These studies show that genetic or chemical reduction of Nte activity results in a neurological phenotype of hyperactivity in mammals and indicate that EOPF toxicity occurs directly through inhibition of Nte without the requirement for Nte gain of function or aging.

Atm haploinsufficiency results in increased sensitivity to sublethal doses of ionizing radiation in mice

Atm haploinsufficiency results in increased sensitivity to sublethal doses of ionizing radiation in mice

Evidence that mouse brain neuropathy target esterase is a lysophospholipase

Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an “NTE-LysoPLA” assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41–45% lower in Nte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.

Expressing what's on your mind: DNA arrays and the brain

Questions about brain function and disease are being addressed with parallel genomic approaches. High-density DNA arrays make it possible to monitor the expression levels of thousands of genes at a time, and are being used to address old questions in new ways and to generate new hypotheses about the workings of the brain.