Kurt Bürki

Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain tau isoform.

Microtubule‐associated protein tau is the major constituent of the paired helical filament, the main fibrous component of the neurofibrillary lesions of Alzheimers disease. Tau is an axonal phosphoprotein in normal adult brain. In Alzheimers disease brain tau is hyperphosphorylated and is found not only in axons, but also in cell bodies and dendrites of affected nerve cells. We report the production and analysis of transgenic mice that express the longest human brain tau isoform under the control of the human Thy‐1 promoter. As in Alzheimers disease, transgenic human tau protein was present in nerve cell bodies, axons and dendrites; moreover, it was phosphorylated at sites that are hyperphosphorylated in paired helical filaments. We conclude that transgenic human tau protein showed pre‐tangle changes similar to those that precede the full neurofibrillary pathology in Alzheimers disease.

Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein

Abstract Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some of these mutations lead to an overproduction of tau isoforms with four microtubule-binding repeats. Here we have expressed the longest four-repeat human brain tau isoform in transgenic mice under the control of the murine Thy1 promoter. Transgenic mice aged 3 weeks to 25 months overexpressed human tau protein in nerve cells of brain and spinal cord. Numerous abnormal, tau-immunoreactive nerve cell bodies and dendrites were seen. In addition, large numbers of pathologically enlarged axons containing neurofilament- and tau-immunoreactive spheroids were present, especially in spinal cord. Signs of Wallerian degeneration and neurogenic muscle atrophy were observed. When motor function was tested, transgenic mice showed signs of muscle weakness. Taken together, these findings demonstrate that overexpression of human four-repeat tau leads to a central and peripheral axonopathy that results in nerve cell dysfunction and amyotrophy.

Aβ is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis

The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis–Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-β40 peptide (Aβ40) to Aβ42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AβDutch40/AβDutch42 toward AβDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Aβ species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Aβ to vascular amyloid pathology and emphasizing the differing roles of Aβ40 and Aβ42 in vascular and parenchymal amyloid pathology.

Dysregulation of Allergic Airway Inflammation in the Absence of Microbial Colonization

RATIONALE The incidence of allergic disorders is increasing in developed countries and has been associated with reduced exposure to microbes and alterations in the commensal bacterial flora. OBJECTIVES To ascertain the relevance of commensal bacteria on the development of an allergic response, we used a model of allergic airway inflammation in germ-free (GF) mice that lack any exposure to pathogenic or nonpathogenic microorganisms. METHODS Allergic airway inflammation was induced in GF, specific pathogen-free (SPF), or recolonized mice by sensitization and challenge with ovalbumin. The resulting cellular infiltrate and cytokine production were measured. MEASUREMENTS AND MAIN RESULTS Our results show that the total number of infiltrating lymphocytes and eosinophils were elevated in the airways of allergic GF mice compared with control SPF mice, and that this increase could be reversed by recolonization of GF mice with the complex commensal flora of SPF mice. Exaggerated airway eosinophilia correlated with increased local production of Th2-associated cytokines, elevated IgE production, and an altered number and phenotype of conventional dendritic cells. Regulatory T-cell populations and regulatory cytokine levels were unaltered, but GF mice exhibited an increased number of basophils and decreased numbers of alveolar macrophages and plasmacytoid dendritic cells. CONCLUSIONS These data demonstrate that the presence of commensal bacteria is critical for ensuring normal cellular maturation, recruitment, and control of allergic airway inflammation.

Establishment of a germ-line competent C57BL/6 embryonic stem cell line.

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.

Mechanisms of Neonatal Mucosal Antibody Protection

Following an abrupt transition at birth from the sterile uterus to an environment with abundant commensal and pathogenic microbes, neonatal mammals are protected by maternal Abs at mucosal surfaces. We show in mice that different Ab isotypes work in distinct ways to protect the neonatal mucosal surface. Secretory IgA acts to limit penetration of commensal intestinal bacteria through the neonatal intestinal epithelium: an apparently primitive process that does not require diversification of the primary natural Ab repertoire. In contrast, neonatal protection against the exclusively luminal parasite Heligmosomoides polygyrus required IgG from primed females. This immune IgG could either be delivered directly in milk or retrotransported via neonatal Fc receptor from the neonatal serum into the intestinal lumen to exert its protective effect.

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa‐reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H‐2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H‐2. RNA analysis and immunofluorescence with TCR V beta‐specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T‐cell‐dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus‐specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.

Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from the inner cell mass of day 3.5 blastocysts of the inbred mouse strain BALB/cJ. Twenty-three lines were karyotyped and three were selected for injection into C57BL/6J host blastocysts. Two of the three lines, BALB/c-I and BALB/c-IV, produced germ-line chimaeras. The suitability of the BALB/c-I line for gene targeting experiments was tested by transfecting a targeting construct for the interleukin-4 (IL-4) gene. Transfected BALB/c-I cells exhibited efficient homologous recombination of the targeting vector and transmitted the induced mutation through the germline. This newly-characterized BALB/c-ES cell line thus provides an alternative to the traditional 129-derived and the recently described C57BL/6 embryonic stem cell lines, and will be useful in disrupting genes involved in the immune system. Furthermore, the genetically pure BALB/c IL-4 deficient mice will aid in studying the role of IL-4 in several infectious disease models in which the BALB/c mouse is a susceptible strain.

Lymphocyte-mediated Cytotoxicity in vitro and in vivo: Mechanisms and Significance

Two classes of lymphocytes are able to exert contact-dependent cytotoxicity: cytotoxic T cells (CTL) react specifically against target cells presenting processed antigenic peptides on self MHC molecules whereas NK cells lyse a variety of target cells without classical restriction by MHC molecules. Although these two effector populations differ in specificity and lineage, the characteristics of their cytotoxic activity and some of their morphological aspects are remarkably similar (Henkart 1985). Because cytotoxicity is thought to be involved in a wide range of immune processes, the mechanisms accounting for lymphocyte-mediated cytotoxicity were studied intensively ever since cell-mediated cytolysis was first described (Govaertz 1960, Brunner et al. 1970). Important steps in the characterisation of cytolytic effector mechanisms were microscopic and microcinematographic studies showing that cytotoxic T cells form reversible conjugates with their target cells and secrete the content of their cytoplasmic granules during this process (Bykovskaja et al. 1978a,b, Matter 1979, Sanderson & Glauert 1979, Granger & Kolb 1968, Thiernesse et al. 1977, Zagury 1982), the isolation of perforin from granules of cytolytic lymphocytes and the demonstration that isolated perforin possess cytolytic activity in vitro (Podack et al. 1985. Young et al. I986a.b.c)., the observation of circular lesions on target cells lysed by lymphocytes (Dourmashkin et al. 1980, Dennert & Podack 1983) and the subsequent cloning of perforin (Shinkai et al. 1988, Lichtenheld et al. 1988) and other granule-associated proteins (Gershenfeld & Weissman 1986, Bleackley et al. 1988, Masson and

Intestinal Bacteria Condition Dendritic Cells to Promote IgA Production

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyers Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-β, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.