Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans.

Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

Label-free 3D-CLEM Using Endogenous Tissue Landmarks.

Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable "flat embedding" preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

Cross-sectional comparison of small animal [18F]-florbetaben amyloid-PET between transgenic AD mouse models.

We aimed to compare [18F]-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer's disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of ß-amyloid with PET results, and undertook ex vivo autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95±0.04 at five months (N = 5) and 1.04±0.03 (p<0.05) at eight months (N = 7) to 1.07±0.04 (p<0.005) at ten months (N = 6), 1.28±0.06 (p<0.001) at 16 months (N = 6) and 1.39±0.09 (p<0.001) at 19 months (N = 6). SUVR was 0.95±0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to ß-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from -22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p<0.001) and PVE-corrected (R = 0.95, p<0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies.

Immunotherapy alleviates amyloid-associated synaptic pathology in an Alzheimer's disease mouse model.

Cognitive decline in Alzheimer's disease is attributed to loss of functional synapses, most likely caused by synaptotoxic, oligomeric forms of amyloid-ß. Many treatment options aim at reducing amyloid-ß levels in the brain, either by decreasing its production or by increasing its clearance. We quantified the effects of immunotherapy directed against oligomeric amyloid-ß in Tg2576 mice, a mouse model of familial Alzheimer's disease. Treatment of 12-month-old mice with oligomer-specific (A-887755) or conformation-unspecific (6G1) antibodies for 8 weeks did not affect fibrillar plaque density or growth. We also quantified densities of DLG4 (previously known as PSD95) expressing post-synapses and synapsin expressing presynapses immunohistochemically. We found that both pre- and post-synapses were strongly reduced in the vicinity of plaques, whereas distant from plaques, in the cortex and hippocampal CA1 field, only post-synapses were reduced. Immunotherapy alleviated this synapse loss. Synapse loss was completely abolished distant from plaques, whereas it was only attenuated in the vicinity of plaques. These results suggest that fibrillar plaques may act as reservoirs for synaptotoxic, oligomeric amyloid-ß and that sequestering oligomers suffices to counteract synaptic pathology. Therefore, cognitive function may be improved by immunotherapy even when the load of fibrillar amyloid remains unchanged.

In vivo imaging reveals sigmoidal growth kinetic of ß-amyloid plaques.

A major neuropathological hallmark of Alzheimer's disease is the deposition of amyloid plaques in the brains of affected individuals. Amyloid plaques mainly consist of fibrillar ß-amyloid, which is a cleavage product of the amyloid precursor protein. The amyloid-cascade-hypothesis postulates Aß accumulation as the central event in initiating a toxic cascade leading to Alzheimer's disease pathology and, ultimately, loss of cognitive function. We studied the kinetics of ß-amyloid deposition in Tg2576 mice, which overexpress human amyloid precursor protein with the Swedish mutation. Utilizing long-term two-photon imaging we were able to observe the entire kinetics of plaque growth in vivo. Essentially, we observed that plaque growth follows a sigmoid-shaped curve comprising a cubic growth phase, followed by saturation. In contrast, plaque density kinetics exhibited an asymptotic progression. Taking into account the fact that a critical concentration of Aß is required to seed new plaques, we can propose the following kinetic model of ß-amyloid deposition in vivo. In the early cubic phase, plaque growth is not limited by Aß concentration and plaque density increases very fast. During the transition phase, plaque density stabilizes whereas plaque volume increases strongly reflecting a robust growth of the plaques. In the late asymptotic phase, Aß peptide production becomes rate-limiting for plaque growth. In conclusion, the present study offers a direct link between in vitro and in vivo studies facilitating the translation of Aß-lowering strategies from laboratory models to patients.

Longitudinal assessment of cerebral ß-amyloid deposition in mice overexpressing Swedish mutant ß-amyloid precursor protein using 18F-florbetaben PET.

UNLABELLED: The progression of ß-amyloid deposition in the brains of mice overexpressing Swedish mutant ß-amyloid precursor protein (APP-Swe), a model of Alzheimer disease (AD), was investigated in a longitudinal PET study using the novel ß-amyloid tracer (18)F-florbetaben. METHODS: Groups of APP-Swe and age-matched wild-type (WT) mice (age range, 10-20 mo) were investigated. Dynamic emission recordings were acquired with a small-animal PET scanner during 90 min after the administration of (18)F-florbetaben (9 MBq, intravenously). After spatial normalization of individual PET recordings to common coordinates for mouse brain, binding potentials (BPND) and standardized uptake value ratios (SUVRs) were calculated relative to the cerebellum. Voxelwise analyses were performed using statistical parametric mapping (SPM). Histochemical analyses and ex vivo autoradiography were ultimately performed in a subset of animals as a gold standard assessment of ß-amyloid plaque load. RESULTS: SUVRs calculated from static recordings during the interval of 30-60 min after tracer injection correlated highly with estimates of BPND based on the entire dynamic emission recordings. (18)F-florbetaben binding did not significantly differ in APP-Swe mice and WT animals at 10 and 13 mo of age. At 16 mo of age, the APP-Swe mice had a significant 7.9% increase (P < 0.01) in cortical (18)F-florbetaben uptake above baseline and at 20 mo there was a 16.6% increase (P < 0.001), whereas WT mice did not show any temporal changes in tracer uptake during the interval of follow-up. Voxelwise SPM analyses revealed the first signs of increased cortical binding at 13 mo and confirmed progressive binding increases in both the frontal and the temporal cortices (P < 0.001 uncorrected) to 20 mo. The SUVR strongly correlated with percentage plaque load (R = 0.95, P < 0.001). CONCLUSION: In the first longitudinal PET study in an AD mouse model using the novel ß-amyloid tracer (18)F-florbetaben, the temporal and spatial progression of amyloidogenesis in the brain of APP-Swe mice were sensitively monitored. This method should afford the means for preclinical testing of novel therapeutic approaches to the treatment of AD.

Amyloid plaque formation precedes dendritic spine loss.

Amyloid-beta plaque deposition represents a major neuropathological hallmark of Alzheimer's disease. While numerous studies have described dendritic spine loss in proximity to plaques, much less is known about the kinetics of these processes. In particular, the question as to whether synapse loss precedes or follows plaque formation remains unanswered. To address this question, and to learn more about the underlying kinetics, we simultaneously imaged amyloid plaque deposition and dendritic spine loss by applying two-photon in vivo microscopy through a cranial window in double transgenic APPPS1 mice. As a result, we first observed that the rate of dendritic spine loss in proximity to plaques is the same in both young and aged animals. However, plaque size only increased significantly in the young cohort, indicating that spine loss persists even many months after initial plaque appearance. Tracking the fate of individual spines revealed that net spine loss is caused by increased spine elimination, with the rate of spine formation remaining constant. Imaging of dendritic spines before and during plaque formation demonstrated that spine loss around plaques commences at least 4 weeks after initial plaque formation. In conclusion, spine loss occurs, shortly but with a significant time delay, after the birth of new plaques, and persists in the vicinity of amyloid plaques over many months. These findings hence give further hope to the possibility that there is a therapeutic window between initial amyloid plaque deposition and the onset of structural damage at spines.

Bis(arylvinyl)pyrazines, -pyrimidines, and -pyridazines as imaging agents for tau fibrils and ß-amyloid plaques in Alzheimer's disease models.

The in vivo diagnosis of Alzheimer's disease (AD) is of high socioeconomic interest and remains a demanding field of research. The biopathological hallmarks of the disease are extracellular plaques consisting of aggregated ß-amyloid peptides (Aß) and tau protein derived intracellular tangles. Here we report the synthesis and evaluation of fluorescent pyrazine, pyrimidine,and pyridazine derivatives in vitro and in vivo aiming at a tau-based diagnosis of AD. The probes were pre-evaluated on human brain tissue by fluorescence microscopy and were found to label all known disease-related alterations at high contrast and specificity. To quantify the binding affinity, a new thiazine red displacement assay was developed and selected candidates were toxicologically profiled. The application in transgenic mouse models demonstrated bioavailability and brain permeability for one compound. In the course of histological testing, we discovered an AD-related deposition of tau aggregates in the Bowman's glands of the olfactory epithelium, which holds potential for an endoscopic diagnosis of AD in the olfactory system.

Long-term in vivo imaging of fibrillar tau in the retina of P301S transgenic mice.

Tauopathies are widespread neurodegenerative disorders characterised by the intracellular accumulation of hyperphosphorylated tau. Especially in Alzheimer's disease, pathological alterations in the retina are discussed as potential biomarkers to improve early diagnosis of the disease. Using mice expressing human mutant P301S tau, we demonstrate for the first time a straightforward optical approach for the in vivo detection of fibrillar tau in the retina. Longitudinal examinations of individual animals revealed the fate of single cells containing fibrillar tau and the progression of tau pathology over several months. This technique is most suitable to monitor therapeutic interventions aimed at reducing the accumulation of fibrillar tau. In order to evaluate if this approach can be translated to human diagnosis, we tried to detect fibrillar protein aggregates in the post-mortem retinas of patients that had suffered from Alzheimer's disease or Progressive Supranuclear Palsy. Even though we could detect hyperphosphorylated tau, we did not observe any fibrillar tau or Aß aggregates. In contradiction to previous studies, our observations do not support the notion that Aß or tau in the retina are of diagnostic value in Alzheimer's disease.

Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells.

PURPOSE: Bevacizumab targets VEGF-A and has proved beneficial in glioma patients, improving clinical symptoms by the reduction of tumor edema. However, it remains controversial whether or not bevacizumab exerts antitumor effects in addition to (and potentially independent of) its effects on tumor vessels, and it is unknown what doses are needed to achieve this. EXPERIMENTAL DESIGN: We established a novel orthotopic glioma mouse model that allowed us to simultaneously study the kinetics of the morphologic and functional vascular changes, tumor growth, and the viability of individual tumor cells during the course of anti-VEGF therapy in the same microscopic tumor region in real-time. Three doses of bevacizumab were compared, a subclinical dose and two clinical doses (medium and high). RESULTS: Low (subclinical) doses of bevacizumab led to a significant reduction of the total vascular volume without affecting tumor cell viability or the overall tumor growth rates. Medium and high doses triggered a similar degree of vascular regression but significantly decreased tumor growth and prolonged survival. Remaining vessels revealed morphologic features of vascular normalization, reduced permeability, and an increase in blood flow velocity; the latter was dose dependent. We observed an uncoupling of the antitumoral and the antivascular effects of bevacizumab with the high dose only, which showed the potential to cause microregional glioma cell regression. In some tumor regions, pronounced glioma cell regression occurred even without vascular regression. In vitro, there was no effect of bevacizumab on glioma cell proliferation. CONCLUSIONS: Regression of glioma cells can occur independently from vascular regression, suggesting that high doses of bevacizumab have indirect anticancer cell properties in vivo.

In vivo multiphoton imaging reveals gradual growth of newborn amyloid plaques over weeks.

The kinetics of amyloid plaque formation and growth as one of the characteristic hallmarks of Alzheimer's disease (AD) are fundamental issues in AD research. Especially the question how fast amyloid plaques grow to their final size after they are born remains controversial. By long-term two-photon in vivo imaging we monitored individual methoxy-X04-stained amyloid plaques over 6 weeks in 12 and 18 months old Tg2576 mice. We found that in 12 months old mice, newly appearing amyloid plaques were initially small in volume and subsequently grew over time. The growth rate of plaques was inversely proportional to their volume; thus amyloid plaques that were already present at the first imaging time point grew over time but slower compared to new plaques. Additionally, we analyzed 18 months old Tg2576 mice in which we neither found newly appearing plaques nor a significant growth of pre-existing plaques over 6 weeks of imaging. In conclusion, newly appearing amyloid plaques are initially small in size but grow over time until plaque growth can not be detected anymore in aged mice. These results suggest that drugs that target plaque formation should be most effective early in the disease, when plaques are growing.

Multiple events lead to dendritic spine loss in triple transgenic Alzheimer's disease mice.

The pathology of Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) peptide, hyperphosphorylated tau protein, neuronal death, and synaptic loss. By means of long-term two-photon in vivo imaging and confocal imaging, we characterized the spatio-temporal pattern of dendritic spine loss for the first time in 3xTg-AD mice. These mice exhibit an early loss of layer III neurons at 4 months of age, at a time when only soluble Aß is abundant. Later on, dendritic spines are lost around amyloid plaques once they appear at 13 months of age. At the same age, we observed spine loss also in areas apart from amyloid plaques. This plaque independent spine loss manifests exclusively at dystrophic dendrites that accumulate both soluble Aß and hyperphosphorylated tau intracellularly. Collectively, our data shows that three spatio-temporally independent events contribute to a net loss of dendritic spines. These events coincided either with the occurrence of intracellular soluble or extracellular fibrillar Aß alone, or the combination of intracellular soluble Aß and hyperphosphorylated tau.

Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease.

Microglia, the immune cells of the brain, can have a beneficial effect in Alzheimer's disease by phagocytosing amyloid-beta. Two-photon in vivo imaging of neuron loss in the intact brain of living Alzheimer's disease mice revealed an involvement of microglia in neuron elimination, indicated by locally increased number and migration velocity of microglia around lost neurons. Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss.

Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway.

Alzheimer's disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the beta-amyloid peptide (Abeta), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Abeta levels and amyloid plaque load via modifying or inhibiting the generation of Abeta. Based on in vivo two-photon imaging, we present evidence that side effects on the level of dendritic spines may counteract the beneficial potential of these approaches. Two potent gamma-secretase inhibitors (GSIs), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) and LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the density of dendritic spines in wild-type mice. In mice deficient for the amyloid precursor protein (APP), both GSIs had no effect on dendritic spine density, demonstrating that gamma-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of gamma-secretase inhibition, we observed a twofold higher density of dendritic spines in the cerebral cortex of adult APP-deficient mice. This observation further supports the notion that APP is involved in the modulation of dendritic spine density--shown here for the first time in vivo.

Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.

Infiltration of cancer cells into normal tissue is a hallmark of malignant gliomas and compromises treatment options. A lack of appropriate models limits the study of this invasion in vivo, which makes it difficult to fully understand its anatomy and the role of dynamic interactions with structures of the normal brain. We developed a novel methodology by utilizing multiphoton laser scanning microscopy (MPLSM) to image the movement of glioma cells deep within the normal brain of live mice in real time. This allowed us to track the invasion of individual RFP-expressing GL261 cells in relation to perfused vasculature or GFP-labeled endothelial cells repetitively over days, up to a depth of 0.5 mm. Glioma cells moved faster and more efficiently when the abluminal site of a blood vessel was utilized for invasion. Cells that invaded perivascularly were frequently found next to (a) multiple capillary structures where microvessels run parallel to each other, (b) capillary loops or glomeruloid-like bodies, and (c) dilated capillaries. Dynamic MPLSM for more than 48 h revealed that single invasive glioma cells induced intussusceptive microvascular growth and capillary loop formation, specifically at the microvascular site with which they had contact. As the main tumor grew by cooption of existing brain vessels, these peritumoral vascular changes may create a beneficial environment for glioma growth. In conclusion, our study revealed new mechanisms of peritumoral angiogenesis and invasion in gliomas, providing an explanation for their interdependence.

Convolution-based one and two component FRAP analysis: theory and application.

The method of fluorescence redistribution after photobleaching (FRAP) is increasingly receiving interest in biological applications as it is nowadays used not only to determine mobility parameters per se, but to investigate dynamic changes in the concentration or distribution of diffusing molecules. Here, we develop a new simple convolution-based approach to analyze FRAP data using the whole image information. This method does not require information about the timing and localization of the bleaching event but uses the first image acquired directly after photobleaching to calculate the intensity distributions, instead. Changes in pools of molecules with different velocities, which are monitored by applying repetitive FRAP experiments within a single cell, can be analyzed by means of a global model by assuming two global diffusion coefficients with changing portions. We validate the approach by simulation and show that translocation of the YFP-fused PH-domain of phospholipase Cdelta1 can be quantitatively monitored by FRAP analysis in a time-resolved manner. The new FRAP data analysis procedure may be applied to investigate signal transduction pathways using biosensors that change their mobility. An altered mobility in response to the activation of signaling cascades may result either from an altered size of the biosensor, e.g. due to multimerization processes or from translocation of the sensor to an environment with different viscosity.

Signal amplification between Gbetagamma release and PI3Kgamma-mediated PI(3,4,5)P3 formation monitored by a fluorescent Gbetagamma biosensor protein and repetitive two component total internal reflection/fluorescence redistribution after photobleaching analysis.

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.