Microglia, the CNS-resident immune cells, play important roles in disease, but the spectrum of their possible activation states is not well understood. We derived co-regulated gene modules from transcriptional profiles of CNS myeloid cells of diverse mouse models, including new tauopathy model datasets. Using these modules to interpret single-cell data from an Alzheimer's disease (AD) model, we identified microglial subsets-distinct from previously reported "disease-associated microglia"-expressing interferon-related or proliferation modules. We then analyzed whole-tissue RNA profiles from human neurodegenerative diseases, including a new AD dataset. Correcting for altered cellular composition of AD tissue, we observed elevated expression of the neurodegeneration-related modules, but also modules not implicated using expression profiles from mouse models alone. We provide a searchable, interactive database for exploring gene expression in all these datasets (http://research-pub.gene.com/BrainMyeloidLandscape). Understanding the dimensions of CNS myeloid cell activation in human disease may reveal opportunities for therapeutic intervention.

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.

The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOHterminal region of Nox1, a protein related to gp91 phox (Nox2) of phagocytic cells, is constitutively associated with ␤Pix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of ␤Pix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, ␤Pix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide (H 2 O 2 ), are produced in mammalian cells in response to the activation of various cell surface receptors and contribute to intracellular signaling and to the regulation of various biological activities, including host defense and metabolic conversion (15,23,35). Receptor-mediated ROS production has been studied extensively in phagocytic cells. The enzyme NADPH oxidase of such cells is composed of at least five protein components, namely two transmembrane flavocytochrome b components (gp91 phox and p22 phox ) and three cytosolic components (p47 phox , p67 phox , and p40 phox ) (2). The exposure of resting phagocytic cells to an appropriate stimulus results in extensive phosphorylation of the cytosolic components of NADPH oxidase and their association with the transmembrane flavocytochrome b components (1,11,33,36). The assembled oxidase complex catalyzes the transfer of an electron to molecular oxygen to yield the superoxide anion, which is then spontaneously or enzymatically converted to H 2 O 2 . The small GTPase Rac is also required for the activation of NADPH oxidase in phagocytic leukocytes (12, 28). Hematopoietic cell-specific Rac2 and the ubiquitously expressed protein Rac1 are the major and minor Rac isoforms, respectively, in human neutrophils (19).Nonphagocytic cells also produce superoxide anions in response to a variety of extracellular stimuli, including plateletderived growth factor (PDGF) and epidermal growth factor (EGF) (3, 5, 35, 38) Several homologs (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) of gp91 phox (Nox2) have been identified in various nonphagocytic cells (8,13,21,23,37). Nox proteins contain binding sites for FAD, NADPH, and heme, and their NH 2 -terminal portions contain a cluster of six hydrophobic segments that are predicted to form transmembrane ␣ helices (23). Some of the gp91 phox homologs likely associate with p22 phox to form functional cytochrome b in nonphagocytes, given that the latter protein is widely expressed (8) and that p22 phox antisense RNA was shown to inhibit angiotensin II-induced ROS gener...

Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion–regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and β-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of β-catenin. β-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and β-catenin promotes excitatory synapse development and function.

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