Tobias Bittner

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 Alzheimers disease by phagocytosing amyloid-β. Two-photon in vivo imaging of neuron loss in the intact brain of living Alzheimers 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.

γ-Secretase Inhibition Reduces Spine Density In Vivo via an Amyloid Precursor Protein-Dependent Pathway

Alzheimers disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the β-amyloid peptide (Aβ), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Aβ levels and amyloid plaque load via modifying or inhibiting the generation of Aβ. 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 γ-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 γ-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of γ-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.

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

The pathology of Alzheimers disease (AD) is characterized by the accumulation of amyloid-β (Aβ) 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 Aβ 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 Aβ 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 Aβ alone, or the combination of intracellular soluble Aβ and hyperphosphorylated tau.

Rescue of Progranulin Deficiency Associated with Frontotemporal Lobar Degeneration by Alkalizing Reagents and Inhibition of Vacuolar ATPase

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR–DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons.

Previous neurophysiological studies on prion protein deficient (Prnp–/–) mice have revealed a significant reduction of slow afterhyperpolarization currents (sIAHP) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrPC. directly affects the potassium channels underlying sIAHP or if sIAHP is indirectly disturbed by altered intracellular Ca2+ fluxes. Patch‐clamp measurements and confocal Ca2+ imaging in acute hippocampal slice preparations of Prnp–/– mice compared to littermate control mice revealed a reduced Ca2+ rise in CA1 neurons lacking PrPC following a depolarization protocol known to induce sIAHP. Moreover, we observed a reduced Ca2+ influx via l‐type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming α1 subunit of VGCCs Prnp–/– mice. Surprisingly, the β2 subunit, critically involved in the transport of the α1 subunit to the plasma membrane, was found to be up‐regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the α1C, D and β2–4 subunits. In conclusion our data support the notion that lack of PrPC. does not directly affect the potassium channels underlying sIAHP, but modulates these channels due to its effect on the intracellular free Ca2+ concentration via a reduced Ca2+ influx through l‐type VGCCs.

Bis(arylvinyl)pyrazines, -pyrimidines, and -pyridazines As Imaging Agents for Tau Fibrils and β-Amyloid Plaques in Alzheimer’s Disease Models

The in vivo diagnosis of Alzheimers 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 β-amyloid peptides (Aβ) 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 Bowmans glands of the olfactory epithelium, which holds potential for an endoscopic diagnosis of AD in the olfactory system.

USE FOR CALIBRATION OF CERTIFIED REFERENCE MATERIALS FOR Aβ1-42

P4-467 LUMIPULSE G TOTALTAU: KEY PERFORMANCES OFA FULLY AUTOMATED CHEMILUMINESCENT IMMUNOASSAY Manu Vandijck, Martine Dauwe, Els Huyck, Nathalie Le Bastard, John Lawson, Christopher Traynham, Zivjena Vucetic, Johan Gobom, Kaj Blennow, Geert Jannes, Vesna Kostanjevecki, Fujirebio Europe N.V., Gent, Belgium; Fujirebio US Inc, Malvern, PA, USA; Fujirebio Diagnostics Inc, Malvern, PA, USA; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden. Contact e-mail: manu. vandijck@fujirebio.com