Literature context: vitrogen Cat# S21374; RRID:AB_2336066 Rabbit anti-Arc P. Worley (Hopk
Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.
Literature context: n Invitrogen: S-21374; 1738253; RRID:AB_2336066 1:100
Perineuronal nets (PNs) are aggregates of extracellular matrix molecules that surround some neurons in the brain. While PNs occur widely across many cortical areas, subcortical PNs are especially associated with motor and auditory systems. The auditory system has recently been suggested as an ideal model system for studying PNs and their functions. However, descriptions of PNs in subcortical auditory areas vary, and it is unclear whether the variation reflects species differences or differences in staining techniques. Here, we used two staining techniques (one lectin stain and one antibody stain) to examine PN distribution in the subcortical auditory system of four different species: guinea pigs (Cavia porcellus), mice (Mus musculus, CBA/CaJ strain), Long-Evans rats (Rattus norvegicus), and naked mole-rats (Heterocephalus glaber). We found that some auditory nuclei exhibit dramatic differences in PN distribution among species while other nuclei have consistent PN distributions. We also found that PNs exhibit molecular heterogeneity, and can stain with either marker individually or with both. PNs within a given nucleus can be heterogeneous or homogenous in their staining patterns. We compared PN staining across the frequency axes of tonotopically organized nuclei and among species with different hearing ranges. PNs were distributed non-uniformly across some nuclei, but only rarely did this appear related to the tonotopic axis. PNs were prominent in all four species; we found no systematic relationship between the hearing range and the number, staining patterns or distribution of PNs in the auditory nuclei.
Literature context: with Alexa Fluor 647 antibody (RRID:AB_2336066; Thermo Fisher Scientific) were
During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.
Literature context: 647 Thermo Fisher Cat# S32357; RRID:AB_2336066 anti-mouse Ly6G clone 1A8, puri
Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer's disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease.
Literature context: S-21374, RRID:AB_2336066) diluted i
The nuclear factor I (NFI) family of transcription factors plays an important role in the development of the cerebral cortex in humans and mice. Disruption of nuclear factor IA (NFIA), nuclear factor IB (NFIB), or nuclear factor IX (NFIX) results in abnormal development of the corpus callosum, lateral ventricles, and hippocampus. However, the expression or function of these genes has not been examined in detail in the adult brain, and the cell type-specific expression of NFIA, NFIB, and NFIX is currently unknown. Here, we demonstrate that the expression of each NFI protein shows a distinct laminar pattern in the adult mouse neocortex and that their cell type-specific expression differs depending on the family member. NFIA expression was more frequently observed in astrocytes and oligodendroglia, whereas NFIB expression was predominantly localized to astrocytes and neurons. NFIX expression was most commonly observed in neurons. The NFI proteins were equally distributed within microglia, and the ependymal cells lining the ventricles of the brain expressed all three proteins. In the hippocampus, the NFI proteins were expressed during all stages of neural stem cell differentiation in the dentate gyrus, with higher expression intensity in neuroblast cells as compared to quiescent stem cells and mature granule neurons. These findings suggest that the NFI proteins may play distinct roles in cell lineage specification or maintenance, and establish the basis for further investigation of their function in the adult brain and their emerging role in disease.
Literature context: # S21374; RRID:AB_2336066 Guinea pig
Proopiomelanocortin (POMC) neurons are critical sensors of nutrient availability implicated in energy balance and glucose metabolism control. However, the precise mechanisms underlying nutrient sensing in POMC neurons remain incompletely understood. We show that mitochondrial dynamics mediated by Mitofusin 1 (MFN1) in POMC neurons couple nutrient sensing with systemic glucose metabolism. Mice lacking MFN1 in POMC neurons exhibited defective mitochondrial architecture remodeling and attenuated hypothalamic gene expression programs during the fast-to-fed transition. This loss of mitochondrial flexibility in POMC neurons bidirectionally altered glucose sensing, causing abnormal glucose homeostasis due to defective insulin secretion by pancreatic β cells. Fed mice lacking MFN1 in POMC neurons displayed enhanced hypothalamic mitochondrial oxygen flux and reactive oxygen species generation. Central delivery of antioxidants was able to normalize the phenotype. Collectively, our data posit MFN1-mediated mitochondrial dynamics in POMC neurons as an intrinsic nutrient-sensing mechanism and unveil an unrecognized link between this subset of neurons and insulin release.
Literature context: onjugate (RRID:AB_2336066). BiP was
Unfavorable redox conditions in the endoplasmic reticulum (ER) can decrease the capacity for protein secretion, altering vital cell functions. While systems to manage reductive stress are well-established, how cells cope with an overly oxidizing ER remains largely undefined. In previous work (Wang et al., 2014), we demonstrated that the chaperone BiP is a sensor of overly oxidizing ER conditions. We showed that modification of a conserved BiP cysteine during stress beneficially alters BiP chaperone activity to cope with suboptimal folding conditions. How this cysteine is reduced to reestablish 'normal' BiP activity post-oxidative stress has remained unknown. Here we demonstrate that BiP's nucleotide exchange factor - Sil1 - can reverse BiP cysteine oxidation. This previously unexpected reductant capacity for yeast Sil1 has potential implications for the human ataxia Marinesco-Sjögren syndrome, where it is interesting to speculate that a disruption in ER redox-signaling (due to genetic defects in SIL1) may influence disease pathology.
Literature context: vitrogen, RRID:AB_2336066) as a tert
Axon targeting during the development of the olfactory system is not always accurate, and numerous axons overextend past the target layer into the deeper layers of the olfactory bulb. To date, the fate of the mis-targeted axons has not been determined. We hypothesized that following overextension, the axons degenerate, and cells within the deeper layers of the olfactory bulb phagocytose the axonal debris. We utilized a line of transgenic mice that expresses ZsGreen fluorescent protein in primary olfactory axons. We found that overextending axons closely followed the filaments of radial glia present in the olfactory bulb during embryonic development. Following overextension into deeper layers of the olfactory bulb, axons degenerated and radial glia responded by phagocytosing the resulting debris. We used in vitro analysis to confirm that the radial glia had phagocytosed debris from olfactory axons. We also investigated whether the fate of overextending axons was altered when the development of the olfactory bulb was perturbed. In mice that lacked Sox10, a transcription factor essential for normal olfactory bulb development, we observed a disruption to the morphology and positioning of radial glia and an accumulation of olfactory axon debris within the bulb. Our results demonstrate that during early development of the olfactory system, radial glia play an important role in removing overextended axons from the deeper layers of the olfactory bulb.