Lisa Garrett

Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA

Huntington disease (HD) is an adult-onset, autosomal dominant inherited human neurodegenerative disorder characterized by hyperkinetic involuntary movements, including motor restlessness and chorea, slowing of voluntary movements and cognitive impairment. Selective regional neuron loss and gliosis in striatum, cerebral cortex, thalamus, subthalamus and hippocampus are well recognized as neuropathological correlates for the clinical manifestations of HD. The underlying genetic mutation is the expansion of CAG trinucleotide repeats (coding for polyglutamines) to 36-121 copies in exon 1 of the HD gene . The HD mRNA and protein product (huntingtin) show widespread distribution, and thus much remains to be understood about the selective and progressive neurodegeneration in HD. To create an experimental animal model for HD, transgenic mice were generated showing widespread expression of full-length human HD cDNA with either 16, 48 or 89 CAG repeats. Only mice with 48 or 89 CAG repeats manifested progressive behavioural and motor dysfunction with neuron loss and gliosis in striatum, cerebral cortex, thalamus and hippocampus. These animals represent clinically relevant models for HD pathogenesis, and may provide insights into the underlying pathophysiological mechanisms of other triplet repeat disorders.

An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene

A pseudogene is a gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain up to 20,000 pseudogenes. Although much effort has been devoted to understanding the function of pseudogenes, their biological roles remain largely unknown. Here we report the role of an expressed pseudogene—regulation of messenger-RNA stability—in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorin1, called Makorin1-p1. This insertion reduced transcription of Makorin1-p1, resulting in destabilization of Makorin1 mRNA in trans by way of a cis-acting RNA decay element within the 5′ region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. Either Makorin1 or Makorin1-p1 transgenes could rescue these phenotypes. Our findings demonstrate a specific regulatory role of an expressed pseudogene, and point to the functional significance of non-coding RNAs.

Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFα was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFα is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFα-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2.

Wnt Signaling Gradients Establish Planar Cell Polarity by Inducing Vangl2 Phosphorylation through Ror2

It is fundamentally important that signaling gradients provide positional information to govern morphogenesis of multicellular organisms. Morphogen gradients can generate different cell types in specific spatial order at distinct threshold concentrations. However, it is largely unknown whether and how signaling gradients also control cell polarities by acting as global cues. Here, we show that Wnt signaling gradient provides directional information to a field of cells. Vangl2, a core component in planar cell polarity, forms Wnt-induced receptor complex with Ror2 to sense Wnt dosages. Wnts dose-dependently induce Vangl2 phosphorylation of serine/threonine residues and Vangl2 activities depend on its levels of phosphorylation. In the limb bud, Wnt5a signaling gradient controls limb elongation by establishing PCP in chondrocytes along the proximal-distal axis through regulating Vangl2 phosphorylation. Our studies have provided new insight to Robinow syndrome, Brachydactyly Type B1, and spinal bifida which are caused by mutations in human ROR2, WNT5A, or VANGL.

The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia.

The fusion gene Cbfb - MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia

Transgenic mice expressing mutant A53T human alpha-synuclein show neuronal dysfunction in the absence of aggregate formation

Alpha-synuclein was implicated in Parkinsons disease when missense mutations in the alpha-synuclein gene were found in autosomal dominant Parkinsons disease and alpha-synuclein was shown to be a major constituent of protein aggregates in sporadic Parkinsons disease and other synucleinopathies. We have generated transgenic mice expressing A53T mutant and wild-type human alpha-synuclein. The mutant transgenic protein was distributed abnormally to the axons, perikarya, and dendrites of neurons in many brain areas. In electron microscopic immunogold studies, no aggregation of alpha-synuclein was found in these mice. However, behavior analysis showed a progressive reduction of spontaneous vertical motor activity in both mutant lines correlating with the dosage of overexpression. In addition, deficits of grip strength, rotarod performance, and gait were observed in homozygous PrPmtB mice. Transgenic animals expressing mutant alpha-synuclein may be a valuable model to assess specific aspects of the pathogenesis of synucleinopathies.

Targeted Disruption of the Methionine Synthase Gene in Mice

ABSTRACT Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.

Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.

Trinucleotide repeats (CGG)22TGG(CGG)43TGG(CGG)21 from the fragile X gene remain stable in transgenic mice

Abstract Unstable premutation alleles in fragile X contain CGG repeats ranging from 34 to about 200. To study the mechanism of formation and the behavior of dynamic mutations, we constructed and cloned 88 trinucleotide repeats including 43 uninterrupted CGGs and injected them into mouse fertilized oocytes. We analyzed 342 transgenic animals obtained from 6 different founders after one to four generations, and found that the repeats remained stable regardless of the sex of the transmitting mouse. Therefore, we may need to consider factors other than trinucleotide repeat length alone to explain CGG instability and create an animal model.

Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.