The proteomic landscape of synaptic diversity across brain regions and cell types.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

An abundance of free regulatory (19S) proteasome particles regulates neuronal synapses.

The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19S particles near synapses. The free neuronal 19S particles bind and deubiquitylate lysine 63-ubiquitin (Lys63-ub), a non-proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys63-ub interactors. Inhibition of the 19S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.

Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research.

Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock-down, knock-out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR-Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA-based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in-depth information, we will provide specific references throughout.

Proteostatic regulation in neuronal compartments.

Neurons continuously adapt to external cues and challenges, including stimulation, plasticity-inducing signals and aging. These adaptations are critical for neuronal physiology and extended survival. Proteostasis is the process by which cells adjust their protein content to achieve the specific protein repertoire necessary for cellular function. Due to their complex morphology and polarized nature, neurons possess unique proteostatic requirements. Proteostatic control in axons and dendrites must be implemented through regulation of protein synthesis and degradation in a decentralized fashion, but at the same time, it requires integration, at least in part, in the soma. Here, we discuss current understanding of neuronal proteostasis, as well as open questions and future directions requiring further exploration.

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

An early cell shape transition drives evolutionary expansion of the human forebrain.

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.

Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development.

Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation. The method builds upon earlier cerebral organoid methodology, with modifications of embryoid body size and shape to increase surface area and slice culture to maintain nutrient and oxygen access to the interior regions of the organoid, enabling long-term culture. We also describe approaches for introducing exogenous plasmid constructs and for sparse cell labeling to image neuronal axon outgrowth and maturation over time. Together, these methods allow for modeling of later events in cortical development, which are important for neurodevelopmental disease modeling. The protocols described can be easily performed by an experimenter with stem cell culture experience and take 2-3 months to complete, with long-term maturation occurring over several months.

Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output.

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.

Probing human brain evolution and development in organoids.

Expansion of the neocortex is thought to underpin the higher cognitive abilities of a number of mammalian lineages, such as cetaceans, elephants, and primates, with humans exhibiting a particularly enlarged and dense cerebral cortex. However, the evolutionary and developmental mechanisms that led to this expansion are not well-understood and limited to correlative observations. Historically, this has been due to technical and ethical limitations owing to the intractability of various species for functional studies. Nonetheless, comparative neuroanatomical observations have revealed probable cell-biological mechanisms that may have driven reorganization of neocortical architecture and cortical expansion. Furthermore, new in vitro neural differentiation technologies are allowing, for the first time, functional studies to test existing hypotheses and identify new mechanisms. Here, we introduce three-dimensional neural organoids as a powerful new tool for these types of evolutionary-neurodevelopmental studies.