Ein genomweiter Screen nach Modifikatoren von Tau-induzierter Neurodegeneration unter Verwendung von RNAi-vermitteltem Gen-Silencing in Drosophila

Intraneuronale Akkumulationen des Tau-Proteins sind ein gemeinsames Merkmal der sogenannten Tauopathien. Frontotemporale Demenz und Parkinsonismus des Chromosoms 17 (FTDP-17) ist eine Tauopathie, die durch verschiedene Mutationen in dem für Tau kodierenden Gen verursacht wird. Die Pathologie von FTDP-17 ist durch intraneuronale Ablagerungen von Tau, den sogenannten Neurofibrillären Bündeln (NFT), charakterisiert. Die wohl bekannteste Tauopathie ist die Alzheimer-Erkrankung (AD), deren zwei neuropathologische Kennzeichen extrazelluläre Amyloid-Plaques und intrazelluläre NFTs sind. Zur Aufklärung der molekularen Mechanismen, die zur Tau-induzierten Neurodegeneration beitragen, wurde ein genomweiter Screen in einem etablierten Drosophila-Modell durchgeführt: eine transgene Fliegelinie, die eine FTDP-17-assoziierte Variante (Tau[R406W]) des Microtubuli-assoziierten Proteins Tau exprimiert. Die Expression von Tau[R406W] im Facettenauge von Drosophila führt zu einer definierten pathologischen Manifestation, dem rauhen Augenphänotyp (REP). Dieser kann dazu genutzt werden, Modifikationen der Tau-induzierten Pathologie zu evaluieren. Der Screen bestand in der jeweiligen Herunterregulierung der Expression aller Drosophila-Gene mit bekanntem humanen Homolog mittels RNAi. Von 7881 untersuchten RNAi-Linien wurden etwa ein Prozent identifiziert, die einen Einfluss auf den Tau-induzierten REP hatten. Zur Identifizierung der Rolle dieser Kandidaten im Krankheitsmechanismus wurden diese zunächst in funktionale Gruppen aufgeteilt. Interessanterweise fanden sich mehrere Gene, die an intrazellulären Transportmechanismen beteiligt sind, darunter Bestandteile des Dynein/Dynactin-Komplexes, der für den retrograden Mikrotubuli-basierten Transport verantwortlich ist. Experimente zur Aufklärung der funktionalen Zusammenhänge zwischen retrogradem Transport und Tau-vermittelter Toxizität weisen darauf hin, dass in diesem Zusammenspiel eine lysosomale Dysfunktion involviert ist. Weitere Experimente identifizierten Kandidaten, die spezifisch für die R406W-Mutation sind und außerdem solche, bei denen der gezeigte Effekt vom Phosphorylierungsstatus von Tau abhängt. Zusammengefasst erbrachte der Screen eine Reihe von bisher zum Großteil unbekannten genetischen Modifikatoren von Tau-induzierter Pathologie. Aus einer Reihe von Ergebnisses lassen sich potentielle Mechanismen der Modifikation ableiten. Dabei weist diese Arbeit vor allem neue Ansätze auf, um die Tau-induzierte Pathologie zu erforschen. Diese Ansätze können für zukünftige Forschungsvorhaben genutzt werden, die am Ende zu der Entwicklung neuer therapeutischer Strategien beitragen können

eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP) measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy

Schematic representations of the two basic calibration concepts.

<p>The fit approach to measure eSIP parameters using a homogenous fluorescent layer (<b>A</b>) reveals the parameters amplitude (<i>A</i>), full width at half maximum (<i>ω</i>_<i>FWHM</i>), the axial position (<i>z</i>₀) and the offset (<i>I</i>₀). The skewness parameter and the Lorentz-Gauss fraction are not shown. If the solution-based sample is used (<b>B</b>) the steepness of the profile can also be expressed by <i>ω</i>_<i>FWHM</i> (compare Eqs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134980#pone.0134980.e005" target="_blank">5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134980#pone.0134980.e011" target="_blank">9</a>.), and instead of the skewness parameter we included the length constant (<i>LC</i>). Example data are shown in grey, and an appropriate fit is shown in black.</p

Gaining information on X and Y from a commercially available calibration sample.

<p>Neither the layer- nor the solution-based eSIP approach provide information about the lateral imaging properties. Utilizing defined fluorescent structures makes it possible to describe aberrations in these dimensions, too. The grid structure on the commercially available Argolight slide was imaged using two excitation wavelength (440 nm and 488 nm) and maximized field of view (<b>A</b>). The analysis of this grid structure revealed strong distortions which can be quantified using second order polynomial fits. To depict this aberration, the second order term (multiplied with 10⁵) is shown next to the corresponding fit (white lines) for the first excitation (<b>B)</b>. The difference in the two excitations is depicted in <b>C</b> as vectors at the position of grid crossings reflecting the direction and the size of the shift (the size of the arrows are multiplied by 100 for visibility).</p

Using section imaging property parameters to optimize microscope system settings.

<p>To estimate the correction collar setting at a Zeiss LSM 510 confocal microscope, a series of eSIP measurements can be used to define the influence on single eSIP parameters. (<b>A</b>) 2d plot of the intensity parameter for correction collar settings of a 40x/1.2 W C-Apochromat (Zeiss) ranging from 0.14 to 0.19 obtained at a wavelength range from 500 nm to 740 nm with pinhole setting 1.0 AU is shown. The data is derived from a 115 x 115 μm centre region of each plane. Although using an Apochromat the emission wavelength dependency is evident in this measurement. However the optimal settings can easily be found. In analogue fashion the influence of the correction collar was analysed based on the <i>ω</i>_<i>FWHM</i> parameter (<b>B</b>). Interestingly, there is no wavelength dependency for this parameter. Analysing the collimator setting in a lux-FRET paradigm with two different excitations at a Zeiss LSM 780 utilizing a 40x/1.2 W C-Apochromat objective revealed different optimal settings in respect to the observed parameter (<b>C and D</b>). In contrast to an optimization according to the maximal intensity (black), we found a different optimal setting, when the axial position difference between first (440 nm) and the second excitation (488 nm) is measured (red). For the collimator setting series we tested the solution-based approach (<b>C</b>) as well as the Argolight calibration slide (<b>D</b>).</p

Moiré artefacts using structured calibration samples.

<p>Example images of maximum projections, taken from a structured non-homogenous layer (Argolight calibration sample). The depicted images show the same field of view of a vertical grid structure (the “homogenous pattern” of the Argolight slide), which were either scanned perpendicular to the grid (<b>A-D</b>) or in a parallel fashion (<b>E-H</b>) with a decreasing pixel size (increasing number of pixels). A Fourier analysis in x direction of the images (<b>I and J</b>) reveals several instances of moiré artefacts (arrow) in addition to the frequency of the grid itself (arrow head). At low resolution (<b>A and E</b>), these artefacts are dominating the images; the grid structure itself becomes invisible.</p

Extended section imaging property charts from layer and solution-based calibration samples.

<p>Comparable eSIP analysis using the same microscopic system with a homogenous fluorescent layer (<b>A</b>) and the solution-based calibration sample (<b>B</b>). Axial profiles (z profile) are shown in 5 different spatial positions (shown as a scheme within the plot). The 'Var over Mean' plot is scaled to maximum frequency in each Intensity bin to account for the different intensity distributions for layer and solution. The conversion factor (<i>CF</i>) is obtained by a linear fit (black line), which is 527.87±0.17 for the layer and 511.02±0.22 for the solution-based approach. The regression coefficient <i>R</i>² is given to describe the goodness of fit. All eSIP parameters are shown as 3 dimensional plots: Intensity (<i>A</i>) in percent of the maximal photon number (<i>p</i>_max), axial position (<i>z</i>₀) in μm, FWHM or steepness (<i>ω</i>_<i>FWHM</i>) in μm, offset (<i>I</i>₀) in digital levels (DL), skewness (<i>s</i>) or length constant (<i>LC</i>) in 1/μm and the Lorentz-Gauss fraction (<i>m</i>_<i>L</i>). Additional scanning parameters: excitation 440 nm with 2% laser power, emission Channel 529 nm centre wavelength, pinhole 0.5 AU, detector gain 700V, 354 x 354 μm field of view with 512 x 512 pixels, pixel dwell time 1.58 μs, and axial spacing is in (A) 0.2 μm and in (B) sequentially 0.1 μm (10 μm around the glass/solution border), 1 μm (for adjacent 45 μm) and 5 μm (for adjacent 200 μm).To give an impression on the computational load: The analysis of the data from (B) took about 3 minutes using a scripting language (MATLAB) on a good equipped office calculator (Intel Core i7 3.2GHz 64 bit system with 32 GB). An analysis of the data from (A) using the SIP approach published by Brakenhoff et al. can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134980#pone.0134980.s002" target="_blank">S2 Fig</a>.</p

Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix

Rewiring of synaptic circuitry pertinent to memory formation has been associated with morphological changes in dendritic spines and with extracellular matrix (ECM) remodeling. Here, we mechanistically link these processes by uncovering a signaling pathway involving the serotonin 5-HT7 receptor (5-HT7R), matrix metalloproteinase 9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as an MMP-9 substrate in neurons) and find that 5-HT7R stimulation increases local MMP-9 activity, triggering dendritic spine remodeling, synaptic pruning, and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. One important physiological consequence of this interaction includes an increase in neuronal outgrowth and elongation of dendritic spines, which might have a positive effect on complex neuronal processes (e.g., reversal learning and neuronal regeneration)

Analysis of polyQ aggregate load.

<p>(<b>A</b>) Exemplified filter retardation analysis to visualize polyQ aggregates. Decreasing amounts of loaded protein derived from fly heads of control (<i>GMR-GAL4</i>, top), <i>GMR>polyQ</i> (middle) or <i>GMR>polyQ</i> in combination with a candidate suppressor (bottom). (<b>B</b>) Densitometric measures of filter retardation analysis. Data depicted as fold change compared to control (<i>GMR>polyQ</i>) for suppressors and enhancers of polyQ-induced toxicity. Independent homogenates (if available) were used for repetitions. In case of none or only one independent repetition n≤2 is indicated. In all other cases, number of independent repetitions is n≥3. Significant changes are indicated * p<0.05; *** p<0.001.</p

Amelioration of Tau pathology and memory deficits by targeting 5-HT7 receptor

International audienceTauopathies comprise a heterogeneous family of neurodegenerative diseases characterized by pathological accumulation of hyperphosphorylated Tau protein. Pathological changes in serotonergic signaling have been associated with tauopathy etiology, but the underlying mechanisms remain poorly understood. Here, we studied the role of the serotonin receptor 7 (5-HT7R), in a mouse model of tauopathy induced by overexpressing the human Tau[R406W] mutant associated with inherited forms of frontotemporal dementia. We showed that the constitutive 5-HT7R activity is required for Tau hyperphosphorylation and formation of highly bundled Tau structures (HBTS) through G-protein-independent, CDK5-dependent mechanism. We also showed that 5-HT7R physically interacts with CDK5. At the systemic level, 5-HT7R-mediated CDK5 activation induces HBTS leading to neuronal death, reduced long-term potentiation (LTP), and impaired memory in mice. Specific blockade of constitutive 5-HT7R activity in neurons that overexpressed Tau[R406W] prevents Tau hyperphosphorylation, aggregation, and neurotoxicity. Moreover, 5-HT7R knockdown in the prefrontal cortex fully abrogates Tau[R406W]-induced LTP deficits and memory impairments. Thus, 5-HT7R/CDK5 signaling emerged as a new, promising target for tauopathy treatments