Vessel ultrastructure in APP23 transgenic mice after passive anti-Aβ immunotherapy and subsequent intracerebral hemorrhage

Passive immunization of amyloid precursor protein (APP) transgenic mice with anti-amyloid beta (Aβ) antibodies was shown to reduce Aβ-deposition in brain and to improve cognition. However, immunotherapy may also be accompanied by a significant increase in the frequency of intracerebral hemorrhages. Because hemorrhages are associated with amyloid-laden vessels, this raises the question whether high concentrations of anti-Aβ antibodies may directly or indirectly lead to a structural destabilization of the vessel wall. To address this point, transmission electron microscopy was performed and the ultrastructure of bleeding and non-bleeding vessels in immunized and non-immunized APP23 transgenic animals was analyzed. To localize bleeding vessels, hemosiderin-positive macrophages were visualized by pre-embedding Perl's Berlin Blue histochemistry. Vessels were analyzed morphologically, anomalies evaluated and quantified. Bleeding vessels were, furthermore, reconstructed in three dimensions to analyze the spatial distribution of amyloid deposits and other pathological changes of the vessel wall. This in-depth morphological analysis revealed that bleeding vessels in immunized as well as in non-immunized APP23 mice were surrounded by a higher number of macrophages compared to non-bleeding vessels in the same animals. However, no differences in the number of macrophages or other structural parameters, such as amyloid deposition, were observed between bleeding vessels of immunized and non-immunized mice. No pathologies which may indicate impending bleeding were observed in the vascular wall of non-bleeding vessels. We conclude, that the increased hemorrhage frequency observed after passive immunization with anti-Aβ antibodies does not lead to overt structural changes in the vessel wall of APP23 transgenic mice. Minor structural alterations of the vessel wall, however, cannot be excluded due to the sample size of our study and the high complexity of the three-dimensional vessel wall ultrastructure

No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

<div><p>Mutations within the <i>LRRK2</i> gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.</p></div

Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons.

<p><b>A-B:</b> Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; <b>C-H:</b> Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. <b>C1-H2:</b> Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.</p

Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.

<p>Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.</p

LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines.

<p><b>A:</b> RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. <b>B:</b> In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. <b>C:</b> Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.</p

Modeling familial Danish dementia in mice supports the concept of the amyloid hypothesis of Alzheimer's disease

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between β-amyloid (Aβ)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Aβ peptides, the lack of codeposition of the two peptides in crosses between ADan- and Aβ-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain

Aβ42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6–8 weeks and the ratio of human amyloid (A)β42 to Aβ40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models

Treatment with the LRRK2-IN-1 kinase inhibitor does not alter adhesion properties in LRRK2 primary human skin fibroblasts.

<p><b>A-B:</b> Western blot analysis of LRRK2 expression in primary skin fibroblasts from healthy subjects (LRRK2) and PD-patients with mutations in the kinase (A) and ROC domain (B) of LRRK2 after treatment with vehicle or 0.1μM LRRK2 IN-1. The MJFF#2 antibody we used recognizes both human and mouse LRRK2. Brain lysates from a Lrrk2 knock-down mouse (KD) and cortex lysate from a non-tg mouse served as negative and positive controls, respectively. One representative fibroblast line per mutation group is shown and 30μg protein was loaded on a 7% acrylamide SDS-gel. <b>C-D:</b> Percentage of adhered fibroblasts with LRRK2 mutations in the kinase (C) and ROC (D) domain at different time points. No significant differences in adhesion capacity were observed between lines. <b>E-F</b>: Percentage of adhered fibroblasts with LRRK2 mutations in the kinase (E) and ROC (F) domain after treatment with vehicle control (0), 0.1μM or 1μM LRRK2-IN-1 for 30 and 120 minutes. No differences were observed in fibroblasts with LRRK2 mutations in the kinase (E) and ROC domain (F). Data represent mean ± SEM; n = 4 independent experiments (C, E) and n = 3 independent experiments (D, F). Healthy-Subjects (LRRK2) = 4 lines; G2019S LRRK2 patients (GS) = 3 lines; N1437S LRRK2 patient (NS) = 2 lines; R1441C LRRK2 patients (RC) = 1 line. (Two-way ANOVA with Repeated Measures).</p