Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

In this work, we find that CD8 T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49CD8 regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 T cells efficiently eliminated pathogenic gliadin-specific CD4 T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIRCD8 T cells, but not CD4 regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49CD8 T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8 T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Brain function requires precise neural circuit assembly during development. Establishing a functional circuit involves multiple coordinated steps ranging from neural cell fate specification to proper matching between pre- and post-synaptic partners. How neuronal lineage and birth timing influence wiring specificity remains an open question. Recent findings suggest that the relationships between lineage, birth timing, and wiring specificity vary in different neuronal circuits. In this review, we summarize our current understanding of the cellular, molecular, and developmental mechanisms linking neuronal lineage and birth timing to wiring specificity in a few specific systems in Drosophila and mice, and review different methods employed to explore these mechanisms.

NAD(+) and NADH play crucial roles in a variety of biological processes including energy metabolism, mitochondrial functions, and gene expression. Multiple studies have indicated that NAD(+) administration can profoundly decrease oxidative cell death as well as ischemic and traumatic brain injury, suggesting NAD(+) metabolism as a promising therapeutic target for cerebral ischemia and head injury. Cumulating evidence has suggested that NAD(+) can produce its protective effects by multiple mechanisms, including preventing mitochondrial alterations, enhancing energy metabolism, preventing virtually all forms of cell death including apoptosis, necrosis and autophagy, inhibiting inflammation, directly increasing antioxidation capacity of cells and tissues, and activating SIRT1. Increasing evidence has also suggested that NADH metabolism is a potential therapeutic target for treating several neurological disorders. A number of studies have further indicated that multiple NAD(+)-dependent enzymes such as sirtuins, polymerase(ADP-ribose) polymerases (PARPs) and CD38 mediate cell death and multiple biological processes. In this article, an overview of the recent findings regarding the roles of NAD(+)/NADH and NAD(+)-dependent enzymes in cell death and ischemic brain injury is provided. These findings have collectively indicated that NAD(+)/NADH and NAD(+)-dependent enzymes play fundamental roles in oxidative stress-induced cell death and ischemic brain injury, which may become promising therapeutic targets for brain ischemia and multiple other neurological disorders.

No abstract available.

Undergraduate researchers are the next-generation scientists. Here, we call for more attention from our community to the proper training of undergraduates in biomedical research laboratories. By dissecting common pitfalls, we suggest how to better mentor undergraduates and prepare them for flourishing careers.

Proteins localized at the cellular interface mediate cell-cell communication and thus control many aspects of physiology in multicellular organisms. Cell-surface proteomics allows biologists to comprehensively identify proteins on the cell surface and survey their dynamics in physiological and pathological conditions. PEELing provides an integrated package and user-centric web service for analyzing cell-surface proteomics data. With a streamlined and automated workflow, PEELing evaluates data quality using curated references, performs cutoff analysis to remove contaminants, connects to databases for functional annotation, and generates data visualizations. Together with chemical and transgenic tools, PEELing completes a pipeline making cell-surface proteomics analysis handy for every lab.

Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.

Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.

The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.