Fibrillar amyloid plaque formation precedes microglial activation.

In Alzheimer's disease (AD), hallmark ß-amyloid deposits are characterized by the presence of activated microglia around them. Despite an extensive characterization of the relation of amyloid plaques with microglia, little is known about the initiation of this interaction. In this study, the detailed investigation of very small plaques in brain slices in AD transgenic mice of the line APP-PS1(dE9) revealed different levels of microglia recruitment. Analysing plaques with a diameter of up to 10 µm we find that only the half are associated with clear morphologically activated microglia. Utilizing in vivo imaging of new appearing amyloid plaques in double-transgenic APP-PS1(dE9)xCX3CR1+/- mice further characterized the dynamic of morphological microglia activation. We observed no correlation of morphological microglia activation and plaque volume or plaque lifetime. Taken together, our results demonstrate a very prominent variation in size as well as in lifetime of new plaques relative to the state of microglia reaction. These observations might question the existing view that amyloid deposits by themselves are sufficient to attract and activate microglia in vivo.

Pharmacological inhibition of BACE1 impairs synaptic plasticity and cognitive functions.

BACKGROUND: BACE1 (beta site amyloid precursor protein cleaving enzyme 1) is the rate limiting protease in amyloid ß production, hence a promising drug target for the treatment of Alzheimer's disease. Inhibition of BACE1, as the major ß-secretase in vivo with multiple substrates, however is likely to have mechanism-based adverse effects. We explored the impact of long-term pharmacological inhibition of BACE1 on dendritic spine dynamics, synaptic functions, and cognitive performance of adult mice. METHODS: Sandwich enzyme-linked immunosorbent assay was used to assess Aß40 levels in brain and plasma after oral administration of BACE1 inhibitors SCH1682496 or LY2811376. In vivo two-photon microscopy of the somatosensory cortex was performed to monitor structural dynamics of dendritic spines while synaptic functions and plasticity were measured via electrophysiological recordings of excitatory postsynaptic currents and hippocampal long-term potentiation in brain slices. Finally, behavioral tests were performed to analyze the impact of pharmacological inhibition of BACE1 on cognitive performance. RESULTS: Dose-dependent decrease of Aß40 levels in vivo confirmed suppression of BACE1 activity by both inhibitors. Prolonged treatment caused a reduction in spine formation of layer V pyramidal neurons, which recovered after withdrawal of inhibitors. Congruently, the rate of spontaneous and miniature excitatory postsynaptic currents in pyramidal neurons and hippocampal long-term potentiation were reduced in animals treated with BACE1 inhibitors. These effects were not detected in Bace1(-/-) mice treated with SCH1682496, confirming BACE1 as the pharmacological target. Described structural and functional changes were associated with cognitive deficits as revealed in behavioral tests. CONCLUSIONS: Our findings indicate important functions to BACE1 in structural and functional synaptic plasticity in the mature brain, with implications for cognition.

Structural dynamics of dendritic spines are influenced by an environmental enrichment: an in vivo imaging study.

Sensory experience alters neuronal circuits, which is believed to form the basis for learning and memory. On a microscopic level, structural changes of the neuronal network are prominently observable as experience-dependent addition and removal of cortical dendritic spines. By environmental enrichment, we here applied broad sensory stimulation to mice and followed the consequences to dendritic spines in the somatosensory cortex utilizing in vivo microscopy. Additionally to apical dendrites of layer V neurons, which are typically analyzed in in vivo imaging experiments, we investigated basal dendrites of layer II/III neurons and describe for the first time experience-dependent alterations on this population of dendrites. On both classes of cortical dendrites, enriched environment-induced substantial changes determined by increases in density and turnover of dendritic spines. Previously established spines were lost after enriched stimulation. A fraction of experience-induced gained spines survived for weeks, which might therefore be functionally integrated into the neuronal network. Furthermore, we observed an increased density of spines that appeared only transiently. Together, we speculate that the cognitive benefits seen in environmental-enriched animals might be a consequence of both, a higher connectivity of the neuronal network due to more established synapses and an enhanced flexibility due to more transient spines.

Involvement of presenilin holoprotein upregulation in calcium dyshomeostasis of Alzheimer's disease.

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ-secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full-length holoprotein forms, in particular familial Alzheimer's disease-causing forms of PS1, result in significantly attenuated calcium release from thapsigargin- and bradykinin-sensitive stores. Interestingly, treatment of HEK293 cells with γ-secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN-2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.

Lack of Pur-alpha alters postnatal brain development and causes megalencephaly.

Pur-alpha (Purα) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. To better understand the role of Purα in the developing and mature brain, we generated Purα-deficient mice, which we were able to raise to the age of six months. Purα(-/-) mice were born with no obvious pathological condition. We obtained convincing evidence that lack of Purα prolongs the postnatal proliferation of neuronal precursor cells both in the hippocampus and in the cerebellum, however, without affecting the overall number of postmitotic neurons. Independent of these findings, we observed alterations in the expression and distribution of the dendritic protein MAP2, the translation of which has been proposed previously to be Purα-dependent. At the age of 2 weeks, Purα(-/-) mice generated a continuous tremor which persisted throughout lifetime. Finally, adult Purα(-/-) mice displayed a megalencephaly and histopathological findings including axonal swellings and hyperphosphorylation of neurofilaments. Our studies underline the importance of Purα in the proliferation of neuronal precursor cells during postnatal brain development and suggest a role for Purα in the regulation of the expression and cellular distribution of dendritic and axonal proteins. Since recent studies implicate a link between Purα and the fragile X tremor/ataxia syndrome, our Purα(-/-) mouse model will provide new opportunities for understanding the mechanisms of neurodegeneration.

Role of APP for dendritic spine formation and stability.

The amyloid precursor protein (APP) is transported in high amounts to the presynaptic endings where its function is still unknown. Several studies indicate that lack of APP or its overexpression affects the number of dendritic spines, the postsynaptic compartment of excitatory synapses. Since synapse loss has been identified as one of the most important structural correlates of cognitive decline in Alzheimer's diseases (AD), the physiological function of APP at synapses, specifically at dendritic spines, has come into focus in AD research. This review intends to give an overview of the very controversial results on APP expression on dendritic spine number in the mouse brain.

Role of presenilin 1 in structural plasticity of cortical dendritic spines in vivo.

Mutations in presenilins are the major cause of familial Alzheimer's disease (FAD), leading to impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Presenilins are the catalytic subunits of γ-secretase, which itself is critically involved in the processing of amyloid precursor protein to release neurotoxic amyloid ß (Aß). Besides Aß generation, there is growing evidence that presenilins play an essential role in the formation and maintenance of synapses. To further elucidate the effect of presenilin1 (PS1) on synapses, we performed longitudinal in vivo two-photon imaging of dendritic spines in the somatosensory cortex of transgenic mice over-expressing either human wild-type PS1 or the FAD-mutated variant A246E (FAD-PS1). Interestingly, the consequences of transgene expression were different in two subtypes of cortical dendrites. On apical layer 5 dendrites, we found an enhanced spine density in both mice over-expressing human wild-type presenilin1 and FAD-PS1, whereas on basal layer 3 dendrites only over-expression of FAD-PS1 increased the spine density. Time-lapse imaging revealed no differences in kinetically distinct classes of dendritic spines nor was the shape of spines affected. Although γ-secretase-dependent processing of synapse-relevant proteins seemed to be unaltered, higher expression levels of ryanodine receptors suggest a modified Ca(2+) homeostasis in PS1 over-expressing mice. However, the conditional depletion of PS1 in single cortical neurons had no observable impact on dendritic spines. In consequence, our results favor the view that PS1 influences dendritic spine plasticity in a gain-of-function but γ-secretase-independent manner.

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.