Designing a Small Fluorescent Inhibitor to Investigate Soluble Epoxide Hydrolase Engagement in Living Cells.

Soluble epoxide hydrolase (sEH) is a promising target for a number of inflammation-related diseases. In addition, inhibition of sEH has been shown to reduce neuroinflammation, which plays a critical role in the development of central nervous system (CNS) diseases such as Alzheimer's disease. In this study, we present the rational design of a small fluorescent sEH inhibitor. Starting from the clinical candidate GSK2256294A, we replaced the triazine moiety with the 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) fluorophore. The resulting fluorescent sEH inhibitor displayed excellent potency in an in vitro enzyme activity assay (IC50 < 2 nM). The developed inhibitor is applicable in a NanoBRET-based assay system suitable for studying sEH target engagement in living cells. Furthermore, the inhibitor can be used to visualize sEH in sEH-transfected HEK293 cells and in primary mouse astrocytes by fluorescence microscopy.

Microglia Phenotypes Converge in Aging and Neurodegenerative Disease.

Microglia, the primary immune cells of the central nervous system, hold a multitude of tasks in order to ensure brain homeostasis and are one of the best predictors of biological age on a cellular level. We and others have shown that these long-lived cells undergo an aging process that impedes their ability to perform some of the most vital homeostatic functions such as immune surveillance, acute injury response, and clearance of debris. Microglia have been described as gradually transitioning from a homeostatic state to an activated state in response to various insults, as well as aging. However, microglia show diverse responses to presented stimuli in the form of acute injury or chronic disease. This complexity is potentially further compounded by the distinct alterations that globally occur in the aging process. In this review, we discuss factors that may contribute to microglial aging, as well as transcriptional microglia alterations that occur in old age. We then compare these distinct phenotypic changes with microglial phenotype in neurodegenerative disease.

Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice.

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

Long-Term In Vivo Imaging of Individual Microglial Cells.

Microglia are morphologically dynamic cells, neatly arranged in an interconnected three-dimensional lattice throughout the brain, constantly surveying the parenchyma, and swiftly responding to a variety of external stimuli. Capturing the dynamics of their morphology, reaction to trauma, pathogens, or endogenous stimuli, and studying changes in their network in their physiological environment requires the use of two-photon microscopy, as well as a precise repositioning strategy. Herein, we describe a robust repeatable localization method, coupled with optimized in vivo two-photon microscopy for long-term imaging of single microglia cells in the mouse brain.

Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia.

Microglia, the brain's immune cells, maintain homeostasis and sense pathological changes by continuously surveying the parenchyma with highly motile large processes. Here, we demonstrate that microglia also use thin actin-dependent filopodia that allow fast nanoscale sensing within discrete regions. Filopodia are distinct from large processes by their size, speed, and regulation mechanism. Increasing cyclic AMP (cAMP) by activating norepinephrine Gs-coupled receptors, applying nitric oxide, or inhibiting phosphodiesterases rapidly increases filopodia but collapses large processes. Alternatively, Gi-coupled P2Y12 receptor activation collapses filopodia but triggers large processes extension with bulbous tips. Similar control of cytoskeletal dynamics and microglial morphology by cAMP is observed in ramified primary microglia, suggesting that filopodia are intrinsically generated sensing structures. Therefore, nanoscale surveillance of brain parenchyma by microglia requires localized cAMP increases that drive filopodia formation. Shifting intracellular cAMP levels controls the polarity of microglial responses to changes in brain homeostasis and alters the scale of immunosurveillance.

Microglia turnover with aging and in an Alzheimer's model via long-term in vivo single-cell imaging.

To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's ß-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.

Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.

Homeostatic and injury-induced microglia behavior in the aging brain.

Microglia cells are essential for brain homeostasis and have essential roles in neurodegenerative diseases. Aging is the main risk factor for most neurodegenerative diseases, and age-related changes in microglia may contribute to the susceptibility of the aging brain to dysfunction and neurodegeneration. We have analyzed morphology and dynamic behavior of neocortical microglia in their physiological environment in young adult (3-month-old), adult (11- to 12-month-old), and aged (26- to 27-month-old) C57BL/6J-Iba1-eGFP mice using in vivo 2-photon microscopy. Results show that surveying microglial cells in the neocortex exhibit age-related soma volume increase, shortening of processes, and loss of homogeneous tissue distribution. Furthermore, microglial process speed significantly decreased with age. While only a small population of microglia showed soma movement in adult mice, the microglia population with soma movement was increased in aged mice. However, in response to tissue injury, the dynamic microglial response was age-dependently diminished. These results provide novel insights into microglial behavior and indicate that microglial dysfunction in the aging brain may contribute to age-related cognitive decline and neurodegenerative diseases.

Comment on "ApoE-directed therapeutics rapidly clear ß-amyloid and reverse deficits in AD mouse models".

Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) reported that bexarotene rapidly reduces ß-amyloid (Aß) levels and plaque burden in two mouse models of Aß deposition in Alzheimer's disease (AD). We now report that, although bexarotene reduces soluble Aß40 levels in one of the mouse models, the drug has no impact on plaque burden in three strains that exhibit Aß amyloidosis.

Repeatable target localization for long-term in vivo imaging of mice with 2-photon microscopy.

Repetitive in vivo imaging in mice has become an indispensable tool for studying dynamic changes in structure and function of the brain. We describe a head fixation system, which allows rapid re-localization of previously imaged regions of interest (ROIs) within the brain. Such ROIs can be automatically relocated and imaged over weeks to months with negligible rotational change and only minor translational errors. Previously stored imaging positions can be fully automated re-localized within a few seconds. This automated rapid and accurate relocation simplifies image acquisition and post-processing in longitudinal imaging experiments. Moreover, as the laser is only used for data acquisition and not for finding previously imaged ROIs, the risk of laser induced tissue damage and photobleaching is greatly reduced. Thus, here described head fixation device appears well suited for in vivo repetitive long-term imaging in rodent brain.

Long-term in vivo imaging of ß-amyloid plaque appearance and growth in a mouse model of cerebral ß-amyloidosis.

Extracellular deposition of the amyloid-ß peptide (Aß) in the brain parenchyma is a hallmark lesion of Alzheimer's disease (AD) and a predictive marker for the progression of preclinical to symptomatic AD. Here, we used multiphoton in vivo imaging to study Aß plaque formation in the brains of 3- to 4-month-old APPPS1 transgenic mice over a period of 6 months. A novel head fixation system provided robust and efficient long-term tracking of single plaques over time. Results revealed an estimated rate of 35 newly formed plaques per cubic millimeter of neocortical volume per week at 4-5 months of age. At later time points (i.e., in the presence of increasing cerebral ß-amyloidosis), the number of newly formed plaques decreased. On average, both newly formed and existing plaques grew at a similar growth rate of 0.3 µm (radius) per week. A solid knowledge of the dynamics of cerebral ß-amyloidosis in mouse models provides a powerful tool to monitor preclinical Aß targeting therapeutic strategies and eases the interpretation of diagnostic amyloid imaging in humans.

Modeling familial Danish dementia in mice supports the concept of the amyloid hypothesis of Alzheimer's disease.

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between beta-amyloid (Abeta)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Abeta peptides, the lack of codeposition of the two peptides in crosses between ADan- and Abeta-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.