Summary Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small molecule probes or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra (EELS) of each lanthanide deposit via energy-filtered transmission electron microscopy (EFTEM). This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the post-synaptic membrane.

Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high-and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensorydeprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. mass spectrometric proteomics | somatosensory cortex | experience-dependent plasticity | synaptic protein dynamics S ensory information is encoded in the brain by activation of specific neuronal circuits that operate via chemical synapses. Important sensory experiences are stored by strengthening activated synapses in the circuit, a process known as "experience-dependent plasticity" (1). When animals are deprived of sensory experience during development, for example, by trimming rodent whiskers from birth to 30 d after birth, synaptic strength and mature synaptic morphology are attenuated, suggesting that the low activity in the deprived circuits interferes with normal development (2-7). However, the molecular changes that alter these synaptic properties in response to experience remain unclear. Synaptic activation has been shown to promote regulated and highly localized effects on synaptic proteins such as local translation, recruitment, and targeted degradation (8-11), and this spatiotemporal control of protein availability is required for long-term plasticity and synaptic maturation, which are fundamental in memory formation and storage and ultimately for an organism's survival (12-14). The need for highly controlled protein availability is highlighted in studies of neurological diseases, which often result from the disruption of protein availability at the synapse (15-17). ...

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an “out” position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.

Protein synthesis is highly regulated throughout nervous system development, plasticity, and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.

Despite great functional diversity, characterization of the ␣/␤-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the ␣/␤-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the ␣/␤-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.The neuroligins (NLGNs) 3 are postsynaptic proteins that associate with cognate presynaptic partners, the neurexins (1-3, ␣ and ␤) (NRXNs) (1, 2), and are essential for selectivity in synaptic function (3). By virtue of a common structure in their extracellular domain, the NLGNs fall in the ␣/␤-hydrolase fold superfamily of proteins. To date, proteins of this family are known to serve three general functions: 1) catalyzing the hydrolysis of ester and amide substrates as with acetylcholinesterase, 2) serving as chaperones for secretion of hormone precursors such as with thyroglobulin (Tg), and 3) mediating heterophilic synaptic adhesion interactions as found for neuroligin. The common structure of the ␣/␤-hydrolase fold domain shared by the family members (4) suggests that despite the different functions, these proteins share common mechanisms of protein folding and processing.In NLGN, the ␣/␤-hydrolase fold domain is crucial for transsynaptic interactions with NRXN. Both the NRXNs and the NLGNs are transmembrane proteins; they facilitate associations with intracellular proteins at synaptic loci in pre-and postsynaptic neurons (5). Genetic aberrations in both the NLGN and the NRXN protein families have been identified in patients with autism spectrum disorders (6 -13). The R451C substitution in the ␣/␤-hydrolase fold domain of NLGN3 was found in two autistic brothers (7), and this mutation has been characterized extensively. Protein derived from R451C NLGN3 cDNA is predomin...

Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKM recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo.Here we investigated PKM stability in rat neurons to better understand its role during information encoding and storage. We used TimeSTAMP reporters to track the synthesis and degradation of PKM as well as a related atypical PKC, PKC. These reporters revealed that both PKM and PKC were upregulated after chemical LTP induction; however, these new PKM copies exhibited more rapid turnover than basally produced PKM, particularly in dendritic spines. In contrast to PKM, new PKC copies exhibited elevated stability. Stable information storage over long periods of time is more challenging the shorter the metabolic lifetime of the candidate molecules.

Recombinant antibodies produced in Chinese hamster ovary (CHO) cells often exhibit a slight yellow-brown color, but the molecular basis for this color has remained elusive. We predicted that the color results from post-translational modifications on the antibody, because colored species were found to coelute with antibody products during size exclusion chromatography. Previously, modification by oxidation and advanced glycation end products (AGEs) had been shown to give rise to colored and fluorescent species whose spectral properties are in agreement with the spectral properties of CHO-derived recombinant antibodies. To test whether these modifications give rise to the color exhibited in our CHO-derived antibodies, we searched for 8 different oxidation and 28 different glycation and AGE modifications by mass spectrometry in a variety of samples exhibiting varying color intensities. Oxidation and glycation modifications correlated weakly with color in a subset of samples, but several AGEs exhibited a strong correlation with product color in all samples tested. This strong correlation with sample color was verified for a specific AGE, carboxymethyllysine, by ELISA, thus validating the mass spectrometry data. These data indicate that AGEs are at least partially responsible for the color seen in CHO-derived recombinant antibodies.