Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD

TCreERT2, a Transgenic Mouse Line for Temporal Control of Cre-Mediated Recombination in Lineages Emerging from the Primitive Streak or Tail Bud

<div><p>The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the <i>T (Brachyury)</i> promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first <i>in vivo</i> tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.</p></div

TCreERT2 recombination dynamics allows measurement of the <i>in</i><i>vivo</i> rate of axis extension and somitogenesis.

<p>A single dose of tamoxifen was injected at 8∶00 AM to dams carrying an E9.0–E9.5 TCreERT2; R26R litter. After the indicated time interval, embryos were harvested and stained for ßGal activity (<b>A–E</b>). The dotted line in all panels marks the border between the anterior PSM and the most caudal somite. After 6 hours, blue cells can just be discerned (<b>A</b>). The intensity of blue increases progressively by 8 hours (<b>B</b>) and 12 hours (<b>C</b>), indicating an increase in the fraction of recombined cells. By 16 hours, the recombined domain has reached the border between the anterior PSM and youngest somite (<b>D</b>). Over the next 8 hours, the four most caudal somites (bracket in <b>E</b>), which together measure approximately 470 µm, are heavily labeled (24-hour point<b>, E</b>) demonstrating that, at this stage, the embryo is extending about one µm/min (470 µm/480 min) and a somite forms about every 2 hours. In the lateral view in <b>E</b> it appears that somites rostral to the bracket are labeled (i.e, there are more than 4 blue somites), but this is an illusion due to viewing a blue labeled neural tube (NT) through translucent, mostly unlabeled, somites. This is demonstrated by a transverse view, shown in panel <b>F</b>, of the embryo in <b>E</b> (white arrowhead <b>E</b> indicates where embryo was sliced).</p

Tamoxifen adminstration induces TCreERT2-mediated recombination specifically in the primitive streak or tail bud.

<p>Embryos of the indicated age carrying either the TCre (<b>A</b>) or TCreERT2 transgenes (<b>B–M</b>) were assayed for activation of the <i>R26R</i> Cre-reporter by staining for ßGal activity (blue color). TCre; <i>R26R</i> control embryos (<b>A</b>) display widespread recombination at E9.5 compared to TCreERT2;<i>R26R</i> embryos, which, 24 hours after induction, have activated R26R only in tissues recently emerged from the primitive streak at E7.5 and E8.5 (<b>B, C</b>) or tailbud at E 9.5–13.5 (<b>D–H</b>) (white arrows in <b>G</b> and <b>H</b>). An uninduced E9.5 TCreERT2;<i>R26R</i> embryo displays almost no blue cells (<b>I</b>) except for two small clusters (<b>I’</b> and <b>I”,</b> which are enlargements of the boxed regions in <b>I;</b> arrows point to individual blue cells. A similar region is circled in <b>F</b>). 48 hours after induction, TCreERT2;<i>R26R</i> embryos from E9.5–E12.5 display a recombined region that extends more rostrally (<b>J–M</b>) than in the case of a 24-hour induction period.</p

TCreERT2-mediated deletion of <i>ß-catenin</i> at different axial levels.

<p>All embryos shown, of the indicated genotype and stage, with or without a 48-hour Tam induction, were stained for expression of the markers <i>Uncx4.1</i> and <i>Msgn1</i>. Both markers were developed with the same color reaction, but they mark mutually exclusive domains: <i>Uncx4.1</i> is expressed only in stripes in the somites and <i>Msgn1</i> is expressed only in the PSM. <b>A–E</b> are lateral views and <b>F–J</b> are either dorsal views (<b>F–H</b>) or magnified views of the caudal end of the embryo immediately above (<b>I, J</b>). TCre-mediated deletion of <i>ß-catenin</i> causes caudal truncation and disorganized somites at E8.5 (<b>A, F</b>). These E8.5 defects are phenocopied in the Tam-induced TCreERT2; <i>ß-catenin ^flox/null</i> embryos (<b>C,H</b>) but not in the uninduced control (<b>B,G</b>), which is similar to a normal TCreERT2; <i>ß-catenin ^flox/wt</i> embryo (insert in <b>B</b>). Tam induction can produce similar somitogenesis defects later in E10.5 TCreERT2; <i>ß-catenin ^flox/null</i> embryo (<b>E, J</b>). The yellow arrow in <b>E</b> indicates the axial level where caudal somite defects begin as indicated by <i>Uncx4.1</i> staining. The red arrow in <b>J</b> points to residual <i>Msgn1</i> expression. Uninduced E10.5 TCreERT2; <i>ß-catenin ^flox/null</i> control embryos (<b>D, I</b>) are similar to TCreERT2; <i>ß-catenin ^flox/wt</i> embryos (insert in <b>D</b>).</p

GPR124 Functions as a WNT7-Specific Coactivator of Canonical β-Catenin Signaling

G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of β-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of β-catenin signaling in brain endothelium. WNT7-stimulated β-catenin signaling was dependent upon GPR124’s intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical β-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barriergenesis

Impaired Recall of Positional Memory following Chemogenetic Disruption of Place Field Stability

The neural network of the temporal lobe is thought to provide a cognitive map of our surroundings. Functional analysis of this network has been hampered by coarse tools that often result in collateral damage to other circuits. We developed a chemogenetic system to temporally control electrical input into the hippocampus. When entorhinal input to the perforant path was acutely silenced, hippocampal firing patterns became destabilized and underwent extensive remapping. We also found that spatial memory acquired prior to neural silencing was impaired by loss of input through the perforant path. Together, our experiments show that manipulation of entorhinal activity destabilizes spatial coding and disrupts spatial memory. Moreover, we introduce a chemogenetic model for non-invasive neuronal silencing that offers multiple advantages over existing strategies in this setting

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

A central challenge in cancer genomics is the systematic identification of single and cooperating tumor suppressor gene mutations driving cellular transformation and tumor progression in the absence of oncogenic driver mutation(s). Multiple in vitro and in vivo gene inactivation screens have enhanced our understanding of the tumor suppressor gene landscape in various cancers. However, these studies are limited to single or combination gene effects, specific organs, or require sensitizing mutations. In this study, we developed and utilized a Sleeping Beauty transposon mutagenesis system that functions only as a gene trap to exclusively inactivate tumor suppressor genes. Using whole body transposon mobilization in wild type mice, we observed that cumulative gene inactivation can drive tumorigenesis of solid cancers. We provide a quantitative landscape of the tumor suppressor genes inactivated in these cancers and show that, despite the absence of oncogenic drivers, these genes converge on key biological pathways and processes associated with cancer hallmarks

Chronic centrosome amplification without tumorigenesis

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase-mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation