Amy Chen

Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality

Heterozygous mutation or deletion of the ß subunit of platelet-activating factor acetylhydrolase (PAFAH1B1, also known as LIS1) in humans is associated with type I lissencephaly, a severe developmental brain disorder thought to result from abnormal neuronal migration. To further understand the function of PAFAH1B1, we produced three different mutant alleles in mouse Pafah1b1. Homozygous null mice die early in embryogenesis soon after implantation. Mice with one inactive allele display cortical, hippocampal and olfactory bulb disorganization resulting from delayed neuronal migration by a cell-autonomous neuronal pathway. Mice with further reduction of Pafah1b1 activity display more severe brain disorganization as well as cerebellar defects. Our results demonstrate an essential, dosage-sensitive neuronal-specific role for Pafah1b1 in neuronal migration throughout the brain, and an essential role in early embryonic development. The phenotypes observed are distinct from those of other mouse mutants with neuronal migration defects, suggesting that Pafah1b1 participates in a novel pathway for neuronal migration.

Cripto is required for correct orientation of the anterior-posterior axis in the mouse embryo

The anterior–posterior axis of the mouse embryo is established by two distinct organizing centres in the anterior visceral endoderm and the distal primitive streak. These organizers induce and pattern the head and trunk respectively, and have been proposed to be localized through coordinate cell movements that rotate a pre-existing proximal–distal axis,. Here we show that correct localization of both head- and trunk-organizing centres requires Cripto,, a putative signalling molecule that is a member of the EGF-CFC gene family,. Before gastrulation, Cripto is asymmetrically expressed in a proximal–distal gradient in the epiblast, and subsequently is expressed in the primitive streak and newly formed embryonic mesoderm. A Cripto null mutation generated by targeted gene disruption results in homozygous Cripto−/− embryos that mostly consist of anterior neuroectoderm and lack posterior structures, thus resembling a head without a trunk. Notably, markers of the head organizer are located at the distal end of the embryo, whereas markers of the primitive streak are absent or localized to the proximal side. Our results indicate that Cripto signalling is essential for the conversion of a proximal–distal asymmetry into an orthogonal anterior–posterior axis.

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.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration

Background Parkinsons disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

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.

Complete Loss of Ndel1 Results in Neuronal Migration Defects and Early Embryonic Lethality

ABSTRACT Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1 −/− mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1 −/− blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1 +/− mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1 cko/− ) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1 cko/+ -Ndel1 +/− mice or Lis1 +/− -Ndel1 +/− mice displayed more severe neuronal migration defects than Lis1 cko/+ -Ndel1 +/ + mice or Lis1 +/− -Ndel1 +/+ mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of β-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.

The Adaptor Protein CIKS/Act1 Is Essential for IL-25-Mediated Allergic Airway Inflammation

IL-17 is the signature cytokine of recently discovered Th type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production, and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (also known as Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses that these two cytokines elicit. We identify CD11c+ macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo.

Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to α-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated α-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19−/− and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the α-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.