Gerda Mitteregger

Synapse Formation and Function Is Modulated by the Amyloid Precursor Protein

The amyloid precursor protein (APP) is critical in the pathogenesis of Alzheimers disease. The question of its normal biological function in neurons, in which it is predominantly located at synapses, is still unclear. Using autaptic cultures of hippocampal neurons, we demonstrate that hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor-mediated EPSCs. The size of the readily releasable synaptic vesicle pool was also increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP, whereas the amplitude of miniature synaptic currents was not found to be altered. Together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunohistochemical analysis revealing an increase of synaptophysin-positive puncta per cultured APP knock-out neuron. In conclusion, lack of APP affects synapse formation and transmission in cultured hippocampal neurons.

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.

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrP(C). Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.

Dendritic Pathology in Prion Disease Starts at the Synaptic Spine

Spine loss represents a common hallmark of neurodegenerative diseases. However, little is known about the underlying mechanisms, especially the relationship between spine elimination and neuritic destruction. We imaged cortical dendrites throughout a neurodegenerative disease using scrapie in mice as a model. Two-photon in vivo imaging over 2 months revealed a linear decrease of spine density. Interestingly, only persistent spines (lifetime ≥8 d) disappeared, whereas the density of transient spines (lifetime ≤4 d) was unaffected. Before spine loss, dendritic varicosities emerged preferentially at sites where spines protrude from the dendrite. These results implicate that the location where the spine protrudes from the dendrite may be particularly vulnerable and that dendritic varicosities may actually cause spine loss.

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.

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.

The role of the octarepeat region in neuroprotective function of the cellular prion protein.

Structural alterations of the cellular prion protein (PrPC) seem to be the core of the pathogenesis of prion diseases. However, the physiological function of PrPC remains an enigma. Cell culture experiments have indicated that PrPC and in particular its N‐terminal octarepeat region together with the phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathways have a fundamental involvement in neuroprotection and oxidative stress reactions. We used wild‐type mice, PrP knockout (Prnp−/−) animals and transgenic mice that lack the octarepeat region (C4/−) and subjected them to controlled ischemia. We identified an increased cleavage and synthesis of PrPC in ischemic brain areas of wild‐type mice compared with sham controls. The infarct size in Prnp−/− animals was increased threefold when compared with wild‐type mice. The infarct size in C4/− animals was identical to Prnp−/− mice, that is, around three times larger than in wild‐type mice. We showed that the PrP in C4/− mice does not functionally rescue the Prnp−/− phenotype; furthermore it is unable to undergo β cleavage, although an increased amount of C1 fragments was found in ischemic brain areas compared with sham controls. We demonstrated that the N‐terminal octarepeat region has a lead function in PrPC physiology and neuroprotection against oxidative stress in vivo.

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.

Cell-free formation of misfolded prion protein with authentic prion infectivity

Prion propagation has been modeled in vitro; however, the low infectious titer of PrPSc thus generated has cast doubt on the “protein-only” hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrPSc and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrPSc provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.