Mutation of Tyrosine Sites in the Human Alpha-Synuclein Gene Induces Neurotoxicity in Transgenic Mice with Soluble Alpha-Synuclein Oligomer Formation

Overexpression of α-synuclein with tyrosine mutated to phenylalanine at position 125 leads to a severe phenotype with motor impairment and neuropathology in Drosophila. Here, we hypothesized that tyrosine mutations would similarly lead to impaired motor performance with neuropathology in a rodent model. In transgenic mice (ASO), tyrosines at positions 125, 133, and 136 in human α-synuclein were mutated to phenylalanine and cloned into a Thy1.2 expression vector, which was used to create transgenic mouse lines on a mixed genetic background TgN(Thy-1-SNCA-YF)4Emfu (YF). The YF mice had a decreased lifespan and displayed a dramatic motor phenotype with paralysis of both hind- and forelegs. Post-translational modification of α-synuclein due to phosphorylation of serine 129 is often seen in inclusions in the brains of patients with α-synucleinopathies. We observed a slight but significant increase in phosphorylation of serine 129 in the cytosol in YF mice compared to age-matched human α-synuclein transgenic mice (ASO). Conversely, significantly decreased phosphorylation of serine 129 was seen in synaptosomes of YF mice that also contained higher amounts of soluble oligomers. YF mice deposited full-length α-synuclein aggregates in neurons widespread in the CNS with the main occurrence in the forebrain structures of the cerebral cortex, the basal ganglia, and limbic structures. Full-length α-synuclein labeling was also prominent in many nuclear regions of the brain stem, deep cerebellar nuclei, and cerebellar cortex. The study shows that the substitution of tyrosines to phenylalanine in α-synuclein at positions 125, 133, and 136 leads to severe toxicity in vivo. An insignificant change upon tyrosine substitution suggests that the phosphorylation of serine 129 is not the cause of the toxicity

ELISA method to detect α-synuclein oligomers in cell and animal models.

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson's disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers

Deregulated Nras expression in knock-in animals harboring a gammaretroviral long terminal repeat at the Nras/Csde1 locus.

To investigate mechanisms and phenotypic effects of insertional mutagenesis by gammaretroviruses, we have developed mouse lines containing a single Akv 1-99 long terminal repeat (LTR) and a floxed PGK/Tn5 neomycin cassette at the Nras proto-oncogene at positions previously identified as viral integration sites in Akv 1-99 induced tumors. The insert did not compromise the embryonic development, however, the cassette had an effect on Nras expression in all tissues analyzed. Cre-mediated excision of the PGK/Tn5 neomycin cassette in two of the lines caused upregulation of Nras. Altogether, the knock-in alleles are characterized by modulation of expression of the target gene from more than ten-fold upregulation to three-fold downregulation and exemplify various mechanisms of deregulation by insertional mutagenesis. LTR knock-in mice may serve as a tool to investigate mechanisms of retroviral insertional mutagenesis and as a way of constitutive or induced modulation of expression of a target gene

Nras overexpression results in granulocytosis, T-cell expansion and early lethality in mice.

NRAS is a proto-oncogene involved in numerous myeloid malignancies. Here, we report on a mouse line bearing a single retroviral long terminal repeat inserted into Nras. This genetic modification resulted in an increased level of wild type Nras mRNA giving the possibility of studying the function and activation of wild type NRAS. Flow cytometry was used to show a variable but significant increase of immature myeloid cells in spleen and thymus, and of T-cells in the spleen. At an age of one week, homozygous mice began to retard compared to their wild type and heterozygous littermates. Two weeks after birth, animals started to progressively lose weight and die before weaning. Heterozygous mice showed a moderate increase of T-cells and granulocytes but survived to adulthood and were fertile. In homozygous and heterozygous mice Gfi1 and Gcsf mRNA levels were upregulated, possibly explaining the increment in immature myeloid cells detected in these mice. The short latency period indicates that Nras overexpression alone is sufficient to cause dose-dependent granulocytosis and T-cell expansion

Gene expression profiling of murine T-cell lymphoblastic lymphoma identifies deregulation of S-phase initiating genes

In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs

<i>Gscf</i> and <i>Gfi1</i> mRNAs were upregulated in young <i>Nras^LTR9S</i> mice.

<p>(A). Q-RT-PCR quantification of <i>Gcsf</i> expression in spleen normalized to <i>Hprt</i> expression. Compared to wt <i>Gcsf</i> was upregulated in <i>LTR/LTR</i> spleen. (B). Q-RT-PCR quantification of <i>Gfi1</i> expression in spleen normalized to <i>Tbp</i> expression. Compared to wt <i>Gfi1</i> was upregulated in <i>LTR/LTR</i> spleen. (C). Q-RT-PCR quantification of <i>RasGRP1</i> expression normalized to <i>Gapdh</i> expression. Compared to wt <i>RasGRP1</i> was upregulated in <i>LTR/LTR</i> spleen. (D). Q-RT-PCR quantification of <i>Sos1</i> expression normalized to <i>Gapdh</i> expression. Compared to wt <i>Sos1</i> was upregulated in <i>LTR/LTR</i> spleen. For A and B three mice of each genotype were analyzed, for C and D, 3 LTR/LTR, 7 LTR/+, and 3+/+ mice were analyzed. Error bars indicate standard deviation.</p

<i>Nras</i> expression in knock­in animals with and without the neomycin selection marker.

<p><i>Nras</i> expression was quantified by qPCR employing two different methods, SYBR green (amplicon covering part of exon 2 and 3) or a TaqMan hydrolysis probe (amplicon covering part of exon 6 and 7). Expression was normalized to that of <i>Tbp</i> or <i>Gapdh</i> depending on the employed strategy (SYBR green or TaqMan probe, respectively) and represented as relative to that of wild type animals. Panels A and B: qPCR and Western analysis of the LTR9S allele. Only +/+ and +/LTR9S animals are included since LTR9S/LTR9S animal die within three weeks. Panels C and D: qPCR and Western Blot analysis of the LTR9AS allele. Paired Student’s t test was used to determine p-values relative to +/+ animals.</p