Small but mighty: the rise of microprotein biology in neuroscience.

The mammalian central nervous system coordinates a network of signaling pathways and cellular interactions, which enable a myriad of complex cognitive and physiological functions. While traditional efforts to understand the molecular basis of brain function have focused on well-characterized proteins, recent advances in high-throughput translatome profiling have revealed a staggering number of proteins translated from non-canonical open reading frames (ncORFs) such as 5' and 3' untranslated regions of annotated proteins, out-of-frame internal ORFs, and previously annotated non-coding RNAs. Of note, microproteins < 100 amino acids (AA) that are translated from such ncORFs have often been neglected due to computational and biochemical challenges. Thousands of putative microproteins have been identified in cell lines and tissues including the brain, with some serving critical biological functions. In this perspective, we highlight the recent discovery of microproteins in the brain and describe several hypotheses that have emerged concerning microprotein function in the developing and mature nervous system.

Developmental dynamics of RNA translation in the human brain.

The precise regulation of gene expression is fundamental to neurodevelopment, plasticity and cognitive function. Although several studies have profiled transcription in the developing human brain, there is a gap in understanding of accompanying translational regulation. In this study, we performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human-specific and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem-cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and, thus, may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.

Neurons that regulate mouse torpor.

The advent of endothermy, which is achieved through the continuous homeostatic regulation of body temperature and metabolism1,2, is a defining feature of mammalian and avian evolution. However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies-including torpor and hibernation-during which their body temperature decreases far below its homeostatic set-point3-5. How homeothermic mammals initiate and regulate these hypothermic states remains largely unknown. Here we show that entry into mouse torpor, a fasting-induced state with a greatly decreased metabolic rate and a body temperature as low as 20 °C6, is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that restimulation of neurons that were activated during a previous bout of torpor is sufficient to initiate the key features of torpor, even in mice that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1-positive cells, the activity of which accurately determines when mice naturally initiate and exit torpor, and the inhibition of which disrupts the natural process of torpor entry, maintenance and arousal. Taken together, our results reveal a specific neuronal population in the mouse hypothalamus that serves as a core regulator of torpor. This work forms a basis for the future exploration of mechanisms and circuitry that regulate extreme hypothermic and hypometabolic states, and enables genetic access to monitor, initiate, manipulate and study these ancient adaptations of homeotherm biology.

A scalable platform for the development of cell-type-specific viral drivers.

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.