Summary Alzheimer’s disease is characterized by the deposition of senile plaques and progressive dementia. The molecular mechanisms that couple plaque deposition to neural system failure, however, are unknown. Using transgenic mouse models of AD together with multiphoton imaging, we measured neuronal calcium in individual neurites and spines in vivo using the genetically-encoded calcium indicator YC3.6. Quantitative imaging revealed elevated [Ca2+]i (calcium overload) in ~20% of neurites in APP mice with cortical plaques, compared to less than 5% in wildtype mice, PS1-mutant mice, or young APP mice (animals without cortical plaques). Calcium overload depended on the existence and proximity to plaques. The downstream consequences included the loss of spino-dendritic calcium compartmentalization (critical for synaptic integration) and a distortion of neuritic morphologies mediated, in part, by the phosphatase calcineurin. Together, these data demonstrate that senile plaques impair neuritic calcium homeostasis in vivo and result in the structural and functional disruption of neuronal networks.

While senile plaques injure neurons focally, the functional response of astrocytes to Alzheimer's disease pathology is unknown. Using multiphoton fluorescence lifetime imaging microscopy in vivo, we quantitatively imaged astrocytic calcium homeostasis in a mouse model of Alzheimer's disease. Resting calcium was globally elevated in the astrocytic network, but was independent of proximity to individual plaques. Time lapse imaging revealed that calcium transients in astrocytes were more frequent, were synchronously coordinated across long distances, and were uncoupled from neuronal activity. Furthermore, rare intercellular calcium waves were observed, but only in mice with amyloid-β plaques, originating near plaques and spreading radially at least 200 µm. Thus, while neurotoxicity is observed near amyloid-β deposits, there exists a more general astrocyte-based network response to focal pathology.Growing evidence supports the hypothesis that in Alzheimer's disease (AD), synapses fail and dendritic spines are lost in the amyloid-β (Aβ) plaque micro-environment through a combination of changes to synaptic drive, calcium overload, and activation of calciumdependent degenerative processes (1-4). Neurons, however, make up only part of the brain's volume, with astrocytes making up the bulk of the remaining volume. Astrocytes form a structurally interconnected network that, in vitro, exhibit unique long-distance signaling properties that might be revealed in vivo only after pathological trauma. The idea that neural network dysfunction and degeneration fully mediates the memory loss in AD also does not reflect the growing in vivo evidence that astrocytes play an important role in cortical circuit function (5-7). In AD, pathological studies of human cases and mouse models have shown that astrocytes surround plaques and might play a critical role in Aβ deposition and clearance (8-10). Given the profound impact of Aβ deposition on nearby neuronal calcium homeostasis and synaptic function, it is reasonable to hypothesize that astrocyte networks would also be perturbed and might contribute to cortical dysfunction (11). We sought to test whether senile plaque deposition would similarly impact astrocyte calcium homeostasis or dynamic signaling in vivo in a mouse model of AD.To answer these questions, we used multiphoton fluorescent lifetime imaging microscopy (FLIM) to measure resting calcium levels in astrocytes of live mice with cortical plaques (12). We multiplexed the fluorescent properties of a small molecule calcium dye, OregonGreen BAPTA-1 AM (OGB), in the same experimental model and for the same group of cells (Fig. 1A); we used OGB both as a relative indicator of astrocytic activity (intensity) and as a quantitative measure of steady-state [Ca]i (lifetime). We used mice that express mutant human Aβ precursor protein (APP, swe) and mutant presenilin 1 (PS1, ΔE9) in neurons. These mutations lead to an increase in Aβ production and plaque deposition beginning at ~4.5 months

BackgroundAmyloid-β oligomers (oAβ) are thought to mediate neurotoxicity in Alzheimer’s disease (AD), and previous studies in AD transgenic mice suggest that calcium dysregulation may contribute to these pathological effects. Even though AD mouse models remain a valuable resource to investigate amyloid neurotoxicity, the concomitant presence of soluble Aβ species, fibrillar Aβ, and fragments of amyloid precursor protein (APP) complicate the interpretation of the phenotypes.MethodTo explore the specific contribution of soluble oligomeric Aβ (oAβ) to calcium dyshomeostasis and synaptic morphological changes, we acutely exposed the healthy mouse brain, at 3 to 6 months of age, to naturally occurring soluble oligomers and investigated their effect on calcium levels using in vivo multiphoton imaging.ResultsWe observed a dramatic increase in the levels of neuronal resting calcium, which was dependent upon extracellular calcium influx and activation of NMDA receptors. Ryanodine receptors, previously implicated in AD models, did not appear to be primarily involved using this experimental setting. We used the high resolution cortical volumes acquired in-vivo to measure the effect on synaptic densities and observed that, while spine density remained stable within the first hour of oAβ exposure, a significant decrease in the number of dendritic spines was observed 24 h post treatment, despite restoration of intraneuronal calcium levels at this time point.ConclusionsThese observations demonstrate a specific effect of oAβ on NMDA-mediated calcium influx, which triggers synaptic collapse in vivo. Moreover, this work leverages a method to quantitatively measure calcium concentration at the level of neuronal processes, cell bodies and single synaptic elements repeatedly and thus can be applicable to testing putative drugs and/or other intervention methodologies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0169-9) contains supplementary material, which is available to authorized users.

Cerebral amyloid angiopathy (CAA), characterized by extracellular β‐amyloid peptide (Aβ) deposits in vessel walls, is present in the majority of cases of Alzheimer’s disease and is a major cause of hemorrhagic stroke. Although the molecular pathways activated by vascular Aβ are poorly understood, extracellular matrix metalloproteinases (MMP) and Aβ‐induced oxidative stress appear to play important roles. We adapted fluorogenic assays for MMP activity and reactive oxygen species generation for use in vivo. Using multiphoton microcopy in APPswe/PS1dE9 and Tg‐2576 transgenic mice, we observed strong associations between MMP activation, oxidative stress, and CAA deposition in leptomeningeal vessels. Antioxidant treatment with α‐phenyl‐N‐tert‐butyl‐nitrone reduced oxidative stress associated with CAA (∼50% reduction) without affecting MMP activation. Conversely, a selection of agents that inhibit MMP by different mechanisms of action, including minocycline, simvastatin, and GM6001, reduced not only CAA‐associated MMP activation (∼30–40% reduction) but also oxidative stress (∼40% reduction). The inhibitors of MMP did not have direct antioxidant effects. Treatment of animals with α‐phenyl‐N‐tert‐butyl‐nitrone or minocycline did not have a significant effect on CAA progression rates. These data suggest a close association between Aβ‐related MMP activation and oxidative stress in vivo and raise the possibility that treatment with MMP inhibitors may have beneficial effects by indirectly reducing the oxidative stress associated with CAA.

Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer's disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantages to using a ratiometric approach, which allows quantitative determinations of calcium that are independent of dye concentration. Taking advantage of the distinct lifetime contrast of the calcium-free and calcium-bound forms of OGB, we used time-domain lifetime measurements to generate calibration curves for OGB lifetime ratios as a function of calcium concentration. In summary, we demonstrate approaches using commercially available tools to measure calcium concentrations in live cells at multiple scales using lifetime contrast. These approaches are broadly applicable to other fluorescent readouts that exhibit lifetime contrast and serve as powerful alternatives to spectral or intensity readouts in multiplexing experiments.

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