Summary Microglia regulate synaptic circuit remodeling and phagocytose synaptic material in the healthy brain; however, the mechanisms directing microglia to engulf specific synapses and avoid others remain unknown. Here, we demonstrate that an innate immune signaling pathway protects synapses from inappropriate removal. The expression patterns of “don’t eat me” signal CD47 and its receptor, SIRPα, correlated with peak pruning in the developing retinogeniculate system, and mice lacking these proteins exhibited increased microglial engulfment of retinogeniculate inputs and reduced synapse numbers in the dorsal lateral geniculate nucleus. CD47-deficient mice also displayed increased functional pruning as measured by electrophysiology. Additionally, CD47 was found to be required for neuronal activity-mediated changes in engulfment, as microglia in CD47 knockout mice failed to display preferential engulfment of less active inputs. Together, these results demonstrate that CD47-SIRPα signaling prevents excess microglial phagocytosis, and show that molecular brakes can be regulated by activity to protect specific inputs.

We have employed nuclear magnetic resonance (NMR) measurements of hydrogen exchange to identify residue‐level conformational changes in hen egg white lysozyme (HEWL) as induced by salt precipitation. Deuterated HEWL was dissolved into a phosphate (H2O) buffer and precipitated at pH 2.1 upon addition of solid KSCN or (ND4)2SO4, allowing isotope labeling of unfolded regions. After 1 h, each precipitate was then dissolved at pH 3.8 to initiate refolding and preserve labeling and subsequently purified for NMR analysis. HEWL precipitated by 1.0 M KSCN exhibited increased hydrogen exchange at 14 residues out of 42 normally well‐protected in the native state. Of the affected residues, 9 were situated in the β‐sheet/loop domain. A similar, though less extensive, effect was observed at 0.2 M KSCN. Precipitation by 1.2 M (ND4)2SO4 resulted in none of the changes detected with KSCN. The popularity of ammonium sulfate as a precipitant is thus supported by this observed preservation of structural integrity. KSCN, in comparison, produced partial unfolding of specific regions in HEWL due most likely to known preferential interactions between −SCN and proteins. The severity of unfolding increased with KSCN concentration such that, at 1.0 M KSCN, almost the entire β‐sheet/loop domain of HEWL was disrupted. Even so, a portion of the HEWL core encompassed by three α‐helices remained intact, possibly facilitating precipitate dissolution. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 144–155, 1998.

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.