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Impairment of nerve conduction is common in neurodegenerative and neuroinflammatory diseases such as multiple sclerosis (MS), and measurement of evoked potentials (visual, motor, or sensory) has been widely used for diagnosis and recently also as a prognostic marker for MS. We used a classical genetic approach to identify novel genes controlling nerve conduction. First, we used quantitative trait mapping in F2 progeny of B10/SJL mice to identify EAE31, a locus controlling latency of motor evoked potentials (MEPs) and clinical onset of experimental autoimmune encephalomyelitis. Then, by combining congenic mapping, in silico haplotype analyses, and comparative genomics we identified inositol polyphosphate-4-phosphatase, type II (Inpp4b) as the quantitative trait gene for EAE31. Sequence variants of Inpp4b (C/A, exon 13; A/C, exon 14) were identified as differing among multiple mouse strains and correlated with individual cortical MEP latency differences. To evaluate the functional relevance of the amino acid exchanges at positions S474R and H548P, we generated transgenic mice carrying the longer-latency allele (Inpp4b(474R/548P)) in the C57BL/6J background. Inpp4b(474R/548P) mice exhibited significantly longer cortical MEP latencies (4.5 +/- 0.22 ms versus 3.7 +/- 0.13 ms; P = 1.04 x 10(-9)), indicating that INPP4B regulates nerve conduction velocity. An association of an INPP4B polymorphism (rs13102150) with MS was observed in German and Spanish MS cohorts (3676 controls and 911 cases) (P = 8.8 x 10(-3)).

The prion consists essentially of PrP(Sc), a misfolded and aggregated conformer of the cellular protein PrP(C). Whereas PrP(C) deficient mice are clinically healthy, expression of PrP(C) variants lacking its central domain (PrP(DeltaCD)), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrP(C) (residues 1-134), or its central domain (residues 90-134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP(DeltaCD) mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP(DeltaCD) in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrP(DeltaCD), ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.

The enumeration of the interaction partners of the cellular prion protein, PrP(C), may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrP(C). When expressed in transgenic mice, PrP(myc) carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrP(C). PrP(myc) antagonized the toxicity of truncated PrP, restored prion infectibility of PrP(C)-deficient mice, and was physically incorporated into PrP(Sc) aggregates, indicating that it possessed all functional characteristics of genuine PrP(C). We then immunopurified myc epitope-containing protein complexes from PrP(myc) transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrP(C) and may represent component of a multiprotein complex. Selected PrP(C) interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, tauopathies, Huntington's disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition.

Within the CNS, the normal form of cellular prion protein (PrP(C)) is expressed on neurons, oligodendrocytes, and astrocytes. The contribution of these cell types to prion replication and pathogenesis is unclear. To assess the role of oligodendrocytes, we expressed PrP(C) under the control of the myelin basic protein (MBP) promoter in mice lacking endogenous PrP(C). PrP(C) was detected in oligodendrocytes and Schwann cells but not in neurons and astrocytes. MBP-PrP mice never developed scrapie after intracerebral, intraperitoneal, or intraocular challenge with scrapie prions. Transgenic brains did not contain protease-resistant prion protein and did not transmit scrapie when inoculated into PrP(C)-overexpressing indicator mice. To investigate whether prion spread within the CNS depends on oligodendrocytic PrP(C), we implanted PrP(C)-overexpressing neuroectodermal grafts into MBP-PrP brains. After intraocular prion inoculation, none of the grafts showed spongiform encephalopathy or prion infectivity. Hence oligodendrocytes do not support cell-autonomous prion replication, establishment of subclinical disease, and neural spread of prions. Prion resistance sets oligodendrocytes aside from both neurons and astrocytes.

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm-egg interaction.

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2(-/-)/APLP1(-/-) and APLP2(-/-)/APP(-/-) mice proved lethal early postnatally. Surprisingly, APLP1(-/-)/APP(-/-) mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1(-/-)/APP(-/-)/APLP2(+/-) mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro.

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrP(C). Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrP(C) is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.

The most popular approach for generating transgenic mammals is the direct injection of transgenes into one pronucleus of a fertilized oocyte. In the past 15 years microinjection has been successfully applied in laboratory as well as in farm animals. The frequency of transgenic founders, although highly different between the species, is efficient enough to render this technique applicable to a wide range of mammals. The expression levels and patterns of a transgene are initially influenced by the construction of the transgene. However, the overall phenotype of a transgenic organism is influenced by several genetic and environmental factors. Due to the features of this technique not all of the genetic factors can be experimentally controlled by the scientist. In this article we will emphasize some peculiarities which have to be taken into account for the successful performance of transgenesis by pronuclear microinjection.

The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/Emu enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by the Lck promoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhancer-albeit at low levels-also failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed.

Attenuated Edmonston measles virus (MV-Edm) is not pathogenic in standard mice. We show here that MV-Edm inoculated via the natural respiratory route has a limited propagation in the lungs of mice with a targeted mutation inactivating the alpha/beta interferon receptor. A high dose of MV-Edm administered intracerebrally is lethal for about half of these mice. To study the consequences of the availability of a high-affinity receptor for MV propagation, we generated alpha/beta interferon-defective mice expressing human CD46 with human-like tissue specificity. Intranasal infection of these mice with MV-Edm resulted in enhanced spread to the lungs and more prominent inflammatory response. Virus replication was also detected in peripheral blood mononuclear cells, the spleen, and the liver. Moreover, intracerebral inoculation of adult animals with low MV-Edm doses caused encephalitis with almost inevitably lethal outcome. We conclude that in mice alpha/beta interferon controls MV infection and that a high-affinity receptor facilitates, but is not strictly required for, MV spread and pathogenesis.

The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.

Udgivelsesdato: 1997-May, To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.

The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).