Neuroimmune Interactions in the Intestine.

Recent advances have contributed to a mechanistic understanding of neuroimmune interactions in the intestine and revealed an essential role of this cross talk for gut homeostasis and modulation of inflammatory and infectious intestinal diseases. In this review, we describe the innervation of the intestine by intrinsic and extrinsic neurons and then focus on the bidirectional communication between neurons and immune cells. First, we highlight the contribution of neuronal subtypes to the development of colitis and discuss the different immune and epithelial cell types that are regulated by neurons via the release of neuropeptides and neurotransmitters. Next, we review the role of intestinal inflammation in the development of visceral hypersensitivity and summarize how inflammatory mediators induce peripheral and central sensitization of gut-innervating sensory neurons. Finally, we outline the importance of immune cells and gut microbiota for the survival and function of different neuronal populations at homeostasis and during bacterial and helminth infection.

The Gut Microbiome as a Regulator of the Neuroimmune Landscape.

The gut microbiome influences many host physiologies, spanning gastrointestinal function, metabolism, immune homeostasis, neuroactivity, and behavior. Many microbial effects on the host are orchestrated by bidirectional interactions between the microbiome and immune system. Imbalances in this dialogue can lead to immune dysfunction and immune-mediated conditions in distal organs including the brain. Dysbiosis of the gut microbiome and dysregulated neuroimmune responses are common comorbidities of neurodevelopmental, neuropsychiatric, and neurological disorders, highlighting the importance of the gut microbiome-neuroimmune axis as a regulator of central nervous system homeostasis. In this review, we discuss recent evidence supporting a role for the gut microbiome in regulating the neuroimmune landscape in health and disease.

Neuroinflammation During RNA Viral Infections.

Neurotropic RNA viruses continue to emerge and are increasingly linked to diseases of the central nervous system (CNS) despite viral clearance. Indeed, the overall mortality of viral encephalitis in immunocompetent individuals is low, suggesting efficient mechanisms of virologic control within the CNS. Both immune and neural cells participate in this process, which requires extensive innate immune signaling between resident and infiltrating cells, including microglia and monocytes, that regulate the effector functions of antiviral T and B cells as they gain access to CNS compartments. While these interactions promote viral clearance via mainly neuroprotective mechanisms, they may also promote neuropathology and, in some cases, induce persistent alterations in CNS physiology and function that manifest as neurologic and psychiatric diseases. This review discusses mechanisms of RNA virus clearance and neurotoxicity during viral encephalitis with a focus on the cytokines essential for immune and neural cell inflammatory responses and interactions. Understanding neuroimmune communications in the setting of viral infections is essential for the development of treatments that augment neuroprotective processes while limiting ongoing immunopathological processes that cause ongoing CNS disease.

Molecular and Functional Neuroscience in Immunity.

The nervous system regulates immunity and inflammation. The molecular detection of pathogen fragments, cytokines, and other immune molecules by sensory neurons generates immunoregulatory responses through efferent autonomic neuron signaling. The functional organization of this neural control is based on principles of reflex regulation. Reflexes involving the vagus nerve and other nerves have been therapeutically explored in models of inflammatory and autoimmune conditions, and recently in clinical settings. The brain integrates neuro-immune communication, and brain function is altered in diseases characterized by peripheral immune dysregulation and inflammation. Here we review the anatomical and molecular basis of the neural interface with immunity, focusing on peripheral neural control of immune functions and the role of the brain in the model of the immunological homunculus. Clinical advances stemming from this knowledge within the framework of bioelectronic medicine are also briefly outlined.

Early rhombic lip Protogenin+ve stem cells in a human-specific neurovascular niche initiate and maintain group 3 medulloblastoma.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Functional sensory circuits built from neurons of two species.

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.

Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes.

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Brain-wide neural activity underlying memory-guided movement.

Behavior relies on activity in structured neural circuits that are distributed across the brain, but most experiments probe neurons in a single area at a time. Using multiple Neuropixels probes, we recorded from multi-regional loops connected to the anterior lateral motor cortex (ALM), a circuit node mediating memory-guided directional licking. Neurons encoding sensory stimuli, choices, and actions were distributed across the brain. However, choice coding was concentrated in the ALM and subcortical areas receiving input from the ALM in an ALM-dependent manner. Diverse orofacial movements were encoded in the hindbrain; midbrain; and, to a lesser extent, forebrain. Choice signals were first detected in the ALM and the midbrain, followed by the thalamus and other brain areas. At movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Spatiotemporal transcriptome atlas reveals the regional specification of the developing human brain.

Different functional regions of brain are fundamental for basic neurophysiological activities. However, the regional specification remains largely unexplored during human brain development. Here, by combining spatial transcriptomics (scStereo-seq) and scRNA-seq, we built a spatiotemporal developmental atlas of multiple human brain regions from 6-23 gestational weeks (GWs). We discovered that, around GW8, radial glia (RG) cells have displayed regional heterogeneity and specific spatial distribution. Interestingly, we found that the regional heterogeneity of RG subtypes contributed to the subsequent neuronal specification. Specifically, two diencephalon-specific subtypes gave rise to glutamatergic and GABAergic neurons, whereas subtypes in ventral midbrain were associated with the dopaminergic neurons. Similar GABAergic neuronal subtypes were shared between neocortex and diencephalon. Additionally, we revealed that cell-cell interactions between oligodendrocyte precursor cells and GABAergic neurons influenced and promoted neuronal development coupled with regional specification. Altogether, this study provides comprehensive insights into the regional specification in the developing human brain.

Dissecting the functional organization of the C. elegans serotonergic system at whole-brain scale.

Serotonin influences many aspects of animal behavior. But how serotonin acts on its diverse receptors across the brain to modulate global activity and behavior is unknown. Here, we examine how serotonin release in C. elegans alters brain-wide activity to induce foraging behaviors, like slow locomotion and increased feeding. Comprehensive genetic analyses identify three core serotonin receptors (MOD-1, SER-4, and LGC-50) that induce slow locomotion upon serotonin release and others (SER-1, SER-5, and SER-7) that interact with them to modulate this behavior. SER-4 induces behavioral responses to sudden increases in serotonin release, whereas MOD-1 induces responses to persistent release. Whole-brain imaging reveals widespread serotonin-associated brain dynamics, spanning many behavioral networks. We map all sites of serotonin receptor expression in the connectome, which, together with synaptic connectivity, helps predict which neurons show serotonin-associated activity. These results reveal how serotonin acts at defined sites across a connectome to modulate brain-wide activity and behavior.

Brain-wide representations of behavior spanning multiple timescales and states in C. elegans.

Changes in an animal's behavior and internal state are accompanied by widespread changes in activity across its brain. However, how neurons across the brain encode behavior and how this is impacted by state is poorly understood. We recorded brain-wide activity and the diverse motor programs of freely moving C. elegans and built probabilistic models that explain how each neuron encodes quantitative behavioral features. By determining the identities of the recorded neurons, we created an atlas of how the defined neuron classes in the C. elegans connectome encode behavior. Many neuron classes have conjunctive representations of multiple behaviors. Moreover, although many neurons encode current motor actions, others integrate recent actions. Changes in behavioral state are accompanied by widespread changes in how neurons encode behavior, and we identify these flexible nodes in the connectome. Our results provide a global map of how the cell types across an animal's brain encode its behavior.

Light modulates glucose metabolism by a retina-hypothalamus-brown adipose tissue axis.

Public health studies indicate that artificial light is a high-risk factor for metabolic disorders. However, the neural mechanism underlying metabolic modulation by light remains elusive. Here, we found that light can acutely decrease glucose tolerance (GT) in mice by activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) innervating the hypothalamic supraoptic nucleus (SON). Vasopressin neurons in the SON project to the paraventricular nucleus, then to the GABAergic neurons in the solitary tract nucleus, and eventually to brown adipose tissue (BAT). Light activation of this neural circuit directly blocks adaptive thermogenesis in BAT, thereby decreasing GT. In humans, light also modulates GT at the temperature where BAT is active. Thus, our work unveils a retina-SON-BAT axis that mediates the effect of light on glucose metabolism, which may explain the connection between artificial light and metabolic dysregulation, suggesting a potential prevention and treatment strategy for managing glucose metabolic disorders.

Choroid plexus mast cells drive tumor-associated hydrocephalus.

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH in vivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

Hormonal gatekeeping via the blood-brain barrier governs caste-specific behavior in ants.

Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.

Molecular and spatial signatures of mouse brain aging at single-cell resolution.

The diversity and complex organization of cells in the brain have hindered systematic characterization of age-related changes in its cellular and molecular architecture, limiting our ability to understand the mechanisms underlying its functional decline during aging. Here, we generated a high-resolution cell atlas of brain aging within the frontal cortex and striatum using spatially resolved single-cell transcriptomics and quantified changes in gene expression and spatial organization of major cell types in these regions over the mouse lifespan. We observed substantially more pronounced changes in cell state, gene expression, and spatial organization of non-neuronal cells over neurons. Our data revealed molecular and spatial signatures of glial and immune cell activation during aging, particularly enriched in the subcortical white matter, and identified both similarities and notable differences in cell-activation patterns induced by aging and systemic inflammatory challenge. These results provide critical insights into age-related decline and inflammation in the brain.

An in vivo neuroimmune organoid model to study human microglia phenotypes.

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, we developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Our data show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, we demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly.