Murine Thy-1 Research Articles

Cryo-EM structures of tau filaments from the brains of mice transgenic for human mutant P301S Tau

Mice transgenic for human mutant P301S tau are widely used as models for human tauopathies. They develop neurodegeneration and abundant filamentous inclusions made of human mutant four-repeat tau. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the brains of Tg2541 and PS19 mice. Both lines express human P301S tau (0N4R for Tg2541 and 1N4R for PS19) on mixed genetic backgrounds and downstream of different promoters (murine Thy1 for Tg2541 and murine Prnp for PS19). The structures of tau filaments from Tg2541 and PS19 mice differ from each other and those of wild-type tau filaments from human brains. The structures of tau filaments from the brains of humans with mutations P301L, P301S or P301T in MAPT are not known. Filaments from the brains of Tg2541 and PS19 mice share a substructure at the junction of repeats 2 and 3, which comprises residues I297-V312 of tau and includes the P301S mutation. The filament core from the brainstem of Tg2541 mice consists of residues K274-H329 of tau and two disconnected protein densities. Two non-proteinaceous densities are also in evidence. The filament core from the cerebral cortex of line PS19 extends from residues G271-P364 of tau. One strong non-proteinaceous density is also present. Unlike the tau filaments from human brains, the sequences following repeat 4 are missing from the cores of tau filaments from the brains of Tg2541 and PS19 mice.

MO066: The Role of Platelet-Derived Growth Factor in Focal Segmental Glomerulosclerosis

Abstract BACKGROUND AND AIMS Focal segmental glomerulosclerosis (FSGS) describes a primary disease of podocytes (podocytopathy) and a secondary consequence of severe or progressive glomerular damage. No specific treatment options exist for FSGS, with many patients progressing to end-stage kidney disease. While podocyte injury is considered to initiate FSGS, parietal epithelial cells (PECs) were shown to be one of the main effector cells forming FSGS. Given the involvement of platelet-derived growth factor (PDGF) in fibrotic processes, we hypothesized that PDGF signaling is involved in the formation and progression of FSGS. METHOD We analyzed the role of PDGF in FSGS development and progression using a murine Thy1.1 trangenic FSGS model, patient kidney biopsies and primary murine PECs. In a second step, we therapeutically treated mice with FSGS with a neutralizing anti-PDGF-B antibody. RESULTS The podocyte-specific Thy1.1 transgenic mice 3 weeks after anti-Thy1.1 antibody injection showed reduced kidney function due to FSGS development. In this model, compared with control mice, PECs were progressively activated (63.3-fold increase in CD44 expression), showed a significantly increased proliferation (14-fold increase in Ki67 expression), followed by glomerular extracellular matrix deposition (2.6-fold increase in collagen 4) and FSGS development. Fluorescence in situ hybridization showed that mRNA of Pdgfb and Pdgfd was elevated in podocytes and Pdgfrb was upregulated in PECs during the time course of the disease. Similarly, in human FSGS biopsies, gene expression of both ligands, PDGFB and PDGFD, was elevated in podocytes and PDGFRB was increased in PECs compared with healthy glomeruli. Stimulation of primary mouse PECs with PDGF-B and PDGF-D in vitro led to significant activation (1.37- and 1.44-fold increase in CD44 expression, respectively), proliferation (1.46- and 1.36-fold increase, respectively), migration and extracellular matrix production (1.40-fold increase in fibronectin after PDGF-D stimulation) which could be inhibited by imatinib or a neutralizing PDGF-B antibody. In mice with FSGS, both a short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation and profibrotic activation. CONCLUSION PDGFR-β and its ligands PDGF-B and PDGF-D were upregulated in experimental and clinical samples with FSGS in PECs and podocytes, respectively, and the inhibition of PDGF-B reduced PEC proliferation and FSGS development. This suggests paracrine cross-talk between injured podocytes and PECs, leading to PECs activation toward a profibrotic (pro-FSGS) phenotype and FSGS development. Thus, PDGF represents a novel molecular pathway in FSGS pathogenesis and potential target for therapy.

Alpha-Synuclein-induced DNA Methylation and Gene Expression in Microglia

Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. Here, we determined the gene expression and DNA methylation changes in microglia induced by aggregate α-syn. Transgenic murine Thy-1 promoter (mThy1)-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-regulated changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data. Microglial DNA isolated from our 3-month cohort had 5315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3742 DMGs and 3766 DEGs in 13-month mThy1-Asyn mice. These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months. We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.

Pathogenic CD8+ Epidermis-Resident Memory T Cells Displace Dendritic Epidermal T Cells in Allergic Dermatitis

The skin is our interface with the outside world, and consequently it is exposed to a wide range of microbes and allergens. Recent studies have indicated that allergen-specific skin-resident memory T (TRM) cells play a role in allergic contact dermatitis (ACD). However, the composition and dynamics of the epidermal T-cell subsets during ACD are not known. Here we show that exposure of the skin to the experimental contact allergen DNFB results in a displacement of the normally occurring dendritic epidermal T cells (DETC) concomitant with an accumulation of epidermal CD8+CD69+CD103+ TRM cells in mice. By studying knockout mice, we provide evidence that CD8+ T cells are required for the displacement of the DETC and that DETC are not required for recruitment of CD8+ TRM cells to the epidermis following allergen exposure. We demonstrate that the magnitude of the allergic reaction correlates with the number of CD8+ epidermal TRM cells, which again correlates with allergen dose and number of allergen exposures. Finally, in an attempt to elucidate why CD8+ epidermal TRM cells persist in the epidermis, we show that CD8+ epidermal TRM cells have a higher proliferative capability and are bioenergetically more stable, displaying a higher spare respiratory capacity than DETC.

Increased Oxidative Stress Exacerbates α-Synuclein Aggregation In Vivo.

Increasing evidence suggests a relationship between oxidative stress and α-synuclein aggregation, the primary pathological hallmark of Parkinson disease (PD). However, a direct causal relationship has not yet been established in vivo in mouse models of PD. Superoxide dismutase 2 (SOD2) is rate limiting in the antioxidant machinery of the mitochondria and even its partial deficiency elevates oxidative stress in mice. Therefore, in order to investigate a possible interaction between oxidative stress and α-synuclein aggregation in vivo, a transgenic model of PD with haplodeficiency for SOD2 was generated on the basis of the well-characterized murine (Thy-1)-h[A30P]-α-synuclein transgenic line. In comparison with littermate controls with full SOD2 capacity, α-synuclein transgenic mice with partial SOD2 deficiency exhibited a significantly more advanced stage of synucleinopathy at 16 months, as demonstrated by higher median PK-PET blot scores (p < 0.01) and a greater amount of truncated α-synuclein in the insoluble fraction of homogenized brains (p < 0.05). These results show that compromising the capacity to scavenge free radicals can exacerbate α-synuclein aggregation, indicating that elevated levels of oxidative stress could modulate the progression of PD.

Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble-enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivoResults: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574-85. ©2018 AACR.

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

BackgroundAdult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.ResultsSurvival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.ConclusionsThe dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.

Axonopathy in an α-Synuclein Transgenic Model of Lewy Body Disease Is Associated with Extensive Accumulation of C-Terminal–Truncated α-Synuclein

Progressive accumulation of α-synuclein (α-syn) in limbic and striatonigral systems is associated with the neurodegenerative processes in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). The murine Thy-1 (mThy1)-α-syn transgenic (tg) model recapitulates aspects of degenerative processes associated with α-syn accumulation in these disorders. Given that axonal and synaptic pathologies are important features of DLB and PD, we sought to investigate the extent and characteristics of these alterations in mThy1-α-syn tg mice and to determine the contribution of α-syn c-terminally cleaved at amino acid 122 (CT α-syn) to these abnormalities. We generated a novel polyclonal antibody (SYN105) against the c-terminally truncated sequence (amino acids 121 to 123) of α-syn (CT α-syn) and performed immunocytochemical and ultrastructural analyses in mThy1-α-syn tg mice. We found abundant clusters of dystrophic neurites in layers 2 to 3 of the neocortex, the stratum lacunosum, the dentate gyrus, and cornu ammonis 3 of the hippocampus, striatum, thalamus, midbrain, and pons. Dystrophic neurites displayed intense immunoreactivity detected with the SYN105 antibody. Double-labeling studies with antibodies to phosphorylated neurofilaments confirmed the axonal location of full-length and CT α-syn. α-Syn immunoreactive dystrophic neurites contained numerous electrodense laminated structures. These results show that neuritic dystrophy is a prominent pathologic feature of the mThy1-α-syn tg model and suggest that CT α-syn might play an important role in the process of axonal damage in these mice as well as in DLB and PD.

N88S seipin mutant transgenic mice develop features of seipinopathy/BSCL2-related motor neuron disease via endoplasmic reticulum stress

Heterozygosity for mutations (N88S and P90L) in the N-glycosylation site of seipin/BSCL2 is associated with the autosomal dominant motor neuron diseases, spastic paraplegia 17 and distal hereditary motor neuropathy type V, referred to as 'seipinopathies'. Previous in vitro studies have shown that seipinopathy-linked mutations result in accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to the unfolded protein response and cell death, suggesting that seipinopathies is closely associated with ER stress. To further understand the molecular pathogenesis of seipinopathies, we generated a transgenic (tg) mouse line expressing the human N88S seipin mutant with the murine Thy-1 promoter to permit analyses of in vivo phenotypic changes. The N88S seipin tg mice develop a progressive spastic motor deficit, reactive gliosis in the spinal cord and neurogenic muscular atrophy, recapitulating the symptomatic and pathological phenotype in patients of seipinopathy. We also found that expression of mutant seipin in mice upregulated the ER stress marker, immunoglobulin-heavy-chain-binding protein, protein disulfide isomerase and X-box binding protein 1, but was not linked to significant neuronal loss in affected tissue, thereby indicating that ER stress is sufficient, while neuronal death is not necessary, for the development of motor phenotypes of seipinopathies. Our findings in the mutant seipin tg mouse provide clues to understand the relationship with ER stress and neurodegeneration, and the seipin tg mouse is a valid tool for the development of novel therapeutic strategies against ER stress-related diseases.

A longitudinal study of behavioral deficits in an AβPP transgenic mouse model of Alzheimer's disease.

Elucidating the age-dependent alterations in transgenic (Tg) mice overexpressing amyloid-β protein precursor (AβPP) is important for understanding the pathogenesis of Alzheimer's disease (AD) and designing experimental therapies. Cross-studies have previously characterized some time-dependent behavioral and pathological alterations in AβPP Tg mice, however, a more comprehensive longitudinal study is needed to fully examine the progressive nature of behavioral deficits in these mice. In order to better understand the age- and gender-dependent progression of behavioral alterations, we performed a longitudinal study wherein Tg mice overexpressing human AβPP751 with the London (V717I) and Swedish (K670M/N671L) mutations under the regulatory control of the neuron specific murine (m)Thy-1 promoter (mThy1-hAβPP751) were behaviorally analyzed at 3 months and then re-tested at 6 and 9 months of age. The results show that there was an age-associated impairment in learning in the water maze task and habituation in the hole-board task. Motor coordination of the mThy1-hAβPP751 Tg mice was well-preserved throughout the investigated life span however, gender-specific deficits were observed in spontaneous activity and thigmotaxis. Neuropathologically, mThy1-hAβPP751 Tg mice displayed a progressive increase in the number of Aβ plaques and mean plaque size in the cortex and hippocampus from 3 to 6 and from 6 to 9 months of age. Taken together, these results indicate that the mThy1-hAβPP751 Tg mice model AD from the early onset of the disease through to later stages, allowing them to be utilized at numerous points during the timeline for drug test designs.

Association between neuropathology and spatial learning in transgenic hAPPSL × hTAU crossbred mice

The neuropathology of Alzheimer's disease (AD) is characterized by extracellular senile plaques, which are the deposits of b-amyloid peptide and intraneuronal neurofibrillary tangles (NFTs), which are composed of aggregates of hyperphosphorylated forms of tau. A new double transgenic mouse line was established that recapitulates both main neuropathological hallmarks of AD. A cross breeding of transgenic mouse lines expressing htau and hAPP with Swedish and London mutation under the control of the murine Thy1 promoter was performed. Our aim was to investigate the association of age dependent neuropathological features with corresponding spatial learning and memory, which was tested in the Morris Water Maze (MWM). At 5-6 months of age, behavior of male and female hAPPSLx htau double transgenic mice and non-transgenic littermates were evaluated in MWM. Histological analysis of brain samples was performed to determine NFTs and b-amyloid plaques. Both double transgenic and non-transgenic littermates were able to learn the spatial navigation task as reflected by the reduction in time and length required to locate the hidden platform. However, the escape latency of double transgenic mice was higher and the swim path longer when compared to litter mates. A significant effect of the genotype was observed between the two groups especially on day 3, as double transgenic mice revealed a significantly worse performance. In the probe trial double transgenic mice showed reduced ability to remember the previous position of the platform as compared to the non-transgenic littermates. The brain histological analysis of 6 months old double transgenic hAPPSL xhtau crossbred mice showed both b-amyloid deposition as well as human tau positive lesioned neurons. This neuropathological feature of double transgenic hAPPSL × htau mice might be responsible for their impaired spatial behavior detected in the MWM. These subsets of neuropathological disturbances as well as the behavioral deficits make hAPPSL × htau crossbreds suitable to investigate drug candidates against AD.

Parietal Epithelial Cells Participate in the Formation of Sclerotic Lesions in Focal Segmental Glomerulosclerosis

The pathogenesis of the development of sclerotic lesions in focal segmental glomerulosclerosis (FSGS) remains unknown. Here, we selectively tagged podocytes or parietal epithelial cells (PECs) to determine whether PECs contribute to sclerosis. In three distinct models of FSGS (5/6-nephrectomy + DOCA-salt; the murine transgenic chronic Thy1.1 model; or the MWF rat) and in human biopsies, the primary injury to induce FSGS associated with focal activation of PECs and the formation of cellular adhesions to the capillary tuft. From this entry site, activated PECs invaded the affected segment of the glomerular tuft and deposited extracellular matrix. Within the affected segment, podocytes were lost and mesangial sclerosis developed within the endocapillary compartment. In conclusion, these results demonstrate that PECs contribute to the development and progression of the sclerotic lesions that define FSGS, but this pathogenesis may be relevant to all etiologies of glomerulosclerosis.

Overexpression of Glutaminyl Cyclase, the Enzyme Responsible for Pyroglutamate Aβ Formation, Induces Behavioral Deficits, and Glutaminyl Cyclase Knock-out Rescues the Behavioral Phenotype in 5XFAD Mice

Pyroglutamate-modified Aβ (AβpE3-42) peptides are gaining considerable attention as potential key players in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity. Overexpressing AβpE3-42 induced a severe neuron loss and neurological phenotype in TBA2 mice. In vitro and in vivo experiments have recently proven that the enzyme glutaminyl cyclase (QC) catalyzes the formation of AβpE3-42. The aim of the present work was to analyze the role of QC in an AD mouse model with abundant AβpE3-42 formation. 5XFAD mice were crossed with transgenic mice expressing human QC (hQC) under the control of the Thy1 promoter. 5XFAD/hQC bigenic mice showed significant elevation in TBS, SDS, and formic acid-soluble AβpE3-42 peptides and aggregation in plaques. In 6-month-old 5XFAD/hQC mice, a significant motor and working memory impairment developed compared with 5XFAD. The contribution of endogenous QC was studied by generating 5XFAD/QC-KO mice (mouse QC knock-out). 5XFAD/QC-KO mice showed a significant rescue of the wild-type mice behavioral phenotype, demonstrating the important contribution of endogenous mouse QC and transgenic overexpressed QC. These data clearly demonstrate that QC is crucial for modulating AβpE3-42 levels in vivo and prove on a genetic base the concept that reduction of QC activity is a promising new therapeutic approach for AD.

Decoy receptor 3 protects non-obese diabetic mice from autoimmune diabetes by regulating dendritic cell maturation and function

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, regulates immune responses through competing with receptors of Fas ligand (FasL), LIGHT and TNF-like molecule 1A (TL1A). We have previously demonstrated that transgenic expression of DcR3 in a β cell-specific manner significantly protects non-obese diabetic (NOD) mice from autoimmune diabetes. In this study, we further investigated the systemic effect of DcR3 in regulating lymphocytes and dendritic cells in NOD mice. Our results demonstrated that both DcR3 plasmid and protein treatments significantly inhibited insulitis and diabetes. Lymphocytes from DcR3.Fc-treated mice revealed less proliferative potential and transferred ameliorated diabetes. By administration of DcR3.Fc in T1 and T2 double transgenic NOD mice expressing human Thy1 or murine Thy1.1 surface marker under IFN-γ or IL-4 promoter control respectively, we observed a remarkable reduction of Th1 and an increase of Th2 immune responses in vivo. Strikingly, in vitro polarization experiments exhibited that not only Th1 but also Th17 cell differentiation was significantly inhibited in splenocytes treated with DcR3.Fc protein. However, this phenomenon was only observed in splenocytes, not in purified CD4 + T cells, suggesting that DcR3-mediated inhibition of Th1 and Th17 differentiation is not T cell-autonomous and maybe through other cell types such as dendritic cells. Finally, our results demonstrated that DcR3 directly modulates the differentiation and maturation of dendritic cells and subsequently regulates the differentiation and effector function of T cells.

A Novel Transgenic Rat Model with a Full Alzheimer's-Like Amyloid Pathology Displays Pre-Plaque Intracellular Amyloid-β-Associated Cognitive Impairment

Alzheimer's disease (AD) is a neurodegenerative pathology in which amyloid-beta (Abeta) peptide accumulates in different brain areas leading to deposition of plaques and a progressive decline of cognitive functions. After a decade in which a number of transgenic (Tg) mouse models mimicking AD-like amyloid-deposition pathology have been successfully generated, few rat models have been reported that develop intracellular and extracellular Abeta accumulation, together with impairment of cognition. The generation of a Tg rat reproducing the full AD-like amyloid pathology has been elusive. Here we describe the generation and characterization of a new transgenic rat line, coded McGill-R-Thy1-APP, developed to express the human amyloid-beta precursor protein (AbetaPP) carrying both the Swedish and Indiana mutations under the control of the murine Thy1.2 promoter. The selected mono-transgenic line displays an extended phase of intraneuronal Abeta accumulation, already apparent at 1 week after birth, which is widespread throughout different cortical areas and the hippocampus (CA1, CA2, CA3, and dentate gyrus). Homozygous Tg animals eventually produce extracellular Abeta deposits and, by 6 months of age, dense, thioflavine S-positive, amyloid plaques are detected, associated with glial activation and surrounding dystrophic neurites. The cognitive functions in transgenic McGill-R-Thy1-APP rats, as assessed using the Morris water maze task, were found already altered as early as at 3 months of age, when no CNS plaques are yet present. The spatial cognitive impairment becomes more prominent in older animals (13 months), where the behavioral performance of Tg rats positively correlates with the levels of soluble Abeta (trimers) measured in the cortex.

HAPP-SL x hTau crossbreds: A mouse model combining two major Alzheimer hallmarks

Amyloid as well as Tau pathology, still the most important hallmarks in case of AD on the cellular level, were not successfully combined in a transgenic mouse model yet. Amyloid and Tau pathology are described to cause neuronal loss due to direct toxicity, increased oxidative stress, mitochondrial impairment, cytoskeletal deterioration apoptosis and other reasons. The crossbreeding experiments were initiated to investigate whether both transgenes synergistically induce the occurrence of AD related pathologic alterations. We crossbred mice overexpressing TAU441 bearing the missense mutations V337M and R406W under the control of the brain specific murine Thy-1 promoter and mice overexpressing human APP carrying the Swedish and London mutations using the same promoter and a C57BL/6 background in both cases. Brain pathology was investigated by immunohistochemical staining with several anti-human PHF, TAU and Amyloid antibodies. Results showed numerous densely packed human TAU-positive lesioned neurons. In addition to that typical amyloid pathology like extracellular Beta-Amyloid depositions were observed. First hints suggesting a correlation between both pathologies were found and are currently further examined. Behavioural evaluations of cognitive functions using Morris Water Maze (MWM) and New Object Recognition (NOR) tests are ongoing. The hAPP-SL x hTau crossbreds were found to effectively simulate AD pathology. The model is expected to be a proper tool to investigate the efficacy of compounds that address the combination of Amyloid and Tau pathology as present in humans.

Reduced brain volumes in mice expressing APP-Austrian mutation but not in mice expressing APP-Swedish–Austrian mutations

We previously described two transgenic mouse lines expressing sub-endogenous levels of the ‘Austrian’ APP-T714I mutation (driven by the prenatally active PDGF-β promoter; APP-Au mice) and showing intraneuronal Aβ pathology and reduced brain volumes on MRI at 12 and 20 months of age. To further investigate whether reduced brain sizes were caused by neurodegeneration or a neurodevelopmental defect, we now measured brain volumes as early as postnatal day 10. At this age, a distinguishable reduction in brain volumes was absent, indicating that brain volume deficits in APP-Au mice are not caused by a neurodevelopmental defect. To further study the association between intraneuronal Aβ and reduced brain volumes, we further generated and analyzed an APP transgenic mouse model expressing both Austrian and Swedish (K670N/M671L) mutations (APP-SwAu mice). APP-Swedish mutation is known to lead to altered APP processing in the secretory pathway, precluding its later processing in endosomal–lysosomal compartments, the site of intraneuronal Aβ accumulation. Also, to have higher levels of transgene expression only after birth, a murine Thy-1 promoter was utilized for APP-SwAu mouse lines. Despite having five times higher transgene APP levels compared to APP-Au mice, APP-SwAu mice showed significantly lower intraneuronal Aβ levels in the absence of reduced brain volumes, suggesting that intraneuronal Aβ accumulation is related to reduced brain volumes in APP-Au mice. These data also provide a first in vivo indication of altered processing of APP-Swedish at sub-endogenous levels, an effect not observed in mouse models expressing the APP-Swedish mutation in high amounts.

P1-094: New transgenic TAU mouse model with cognitive disturbances

Tauopathies are represented by fronto–temporal dementia, with Parkinsonism linked to chromosome 17 (FTDP–17), amyotrophic lateral sclerosis/parkinsonism–dementia complex, Alzheimer's disease and progressive supranuclear palsy. These diseases are characterized by numerous inclusions, formed by paired helical filaments (PHFs) or filaments composed of hyper–phosphorylated TAU proteins in many brain regions. Several mutations of TAU have been described. Some missense mutations (G272V – exon 9, V337M – exon 12 and R406W – exon 13) affect all isoforms produced, while for example P301L only alters those isoforms with 4 repeats. The R406W tau mutation found in FTDP–17 causes a hereditary tauopathy clinically resembling Alzheimer's disease. The mutation V337M leads to a loss of function of the tau isoforms and provokes microtubule destabilization and tau aggregation. We generated transgenic mice overexpressing human TAU441 gene with two mutations, V337M and R406W, under the control of a tissue specific murine Thy1–promoter. As genetic background C57Bl/6J X CB6 inbred mice were used. From 7 founders offsprings were investigated in terms of TAU RNA and protein content in specific brain regions. Finally 3 different lines with high TAU expression were further characterized. The expanded phenotyping comprised evaluation of cognitive changes, of motor function and especially brain morphology. None of the investigated animals showed signs of motoric disturbances up to an age of 14 months. These results are supported by Rotarod experiments in 9 month old animals showing no disturbance at all. In two out of three lines spatial navigation behaviour in the MWM is severely disturbed compared to littermates. The histological evaluation of TAU pathology in the cortex using the AT180 antibody which detects PHF and tangles (Thr231) revealed numerous densely packed TAU–positive lesions i.e. abnormal TAU–immunoreactive neuropil fibers and tangles. Massive TAU depositions can be seen in neuronal soma and axon, the latter densely packed with TAU. In the hippocampus accentuated TAU accumulation is visible, apparently seen in the periventricular matter. Further investigations will mainly focus on neuronal cell death, the final step in tauopathies like Alzheimer's disease. So far these TAU mouse lines exhibit promising features, relevant for drug testing.

Kinetics of adaptive immunity to Haemophilus influenzae type b meningitis in transgenic mice: Evidence from diverse expression of double T1/T2 transgenes and Th1/Th2-related cytokines

To investigate the kinetic changes in adaptive immunity during experimental Haemophilus influenzae type b (Hib) meningitis, we established a murine meningitis model based on T1/T2 doubly transgenic mice. These mice carry two transgenes that express two distinct cell-surface markers: a human Thy1 transgene ( hThy1) under the control of the murine IFN-γ promoter, and a murine Thy1.1 transgene ( mThy1.1) under the control of the murine IL-4 promoter, designated T1 and T2, respectively. Mice infected with Hib displayed severest symptoms and lowest total splenocyte counts on day 3 after infection. Simultaneously, we examined the significantly low percentage of CD19 + B cells, the relatively high level of CD4 + T cells and significantly high percentage of CD8 + T cells in Hib-infected mice. Furthermore, we observed the early induction of both Th1 and Th2 responses, in terms of the augmentation of Th1 cells (IFN-γ-producing CD4 + T cells) and Th2 cells (IL-4-producing CD4 + T cells) in Hib-infected mice. On day 7 after infection, the Th1 response gradually declined and the Th2 response rather sustained. Two weeks after infection, both Th1 and Th2 cells were barely detectable. Moreover, we demonstrated using an antigen-specific re-stimulation test to analyze the effector function of lymphocyte subsets that CD8 + T cells contributed to more predominantly production of IFN-γ than CD4 + T cells did; and CD4 + T cells partly contributed to the secretion of IL-4 from flowcytometry of intracellular cytokine staining. Our results support that these transgenic mice provide an available model to dissect the complex kinetic change of adaptive immunity in bacterial infectious diseases.

Lack of ABCA1 Considerably Decreases Brain ApoE Level and Increases Amyloid Deposition in APP23 Mice

ABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Abeta immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Abeta fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble beta-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD.