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Identifying gene expression programs underlying both cell-type identity and cellular activities (e.g. life-cycle processes, responses to environmental cues) is crucial for understanding the organization of cells and tissues. Although single-cell RNA-Seq (scRNA-Seq) can quantify transcripts in individual cells, each cell’s expression profile may be a mixture of both types of programs, making them difficult to disentangle. Here, we benchmark and enhance the use of matrix factorization to solve this problem. We show with simulations that a method we call consensus non-negative matrix factorization (cNMF) accurately infers identity and activity programs, including their relative contributions in each cell. To illustrate the insights this approach enables, we apply it to published brain organoid and visual cortex scRNA-Seq datasets; cNMF refines cell types and identifies both expected (e.g. cell cycle and hypoxia) and novel activity programs, including programs that may underlie a neurosecretory phenotype and synaptogenesis.

Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1−/− and Cx3cl1−/− synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.

Behavioural experiences activate the FOS transcription factor in sparse populations of neurons that are critical for encoding and recalling specific events 1 – 3 . However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos -activated cells. It is also not known whether FOS is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos -activated hippocampal CA1 pyramidal neurons by parvalbumin-expressing interneurons is enhanced, whereas perisomatic inhibition by cholecystokinin-expressing interneurons is weakened. This bidirectional modulation of inhibition is abolished when the function of the FOS transcription factor complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling and chromatin analyses, combined with electrophysiology, reveal that FOS activates the transcription of Scg2 , a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As parvalbumin- and cholecystokinin-expressing interneurons mediate distinct features of pyramidal cell activity 4 – 6 , the SCG2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo. Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to theta phase are significantly altered in the absence of Scg2 . These findings reveal an instructive role for FOS and SCG2 in establishing a network of Fos -activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms acting on distinct inhibitory pathways may support the consolidation of memories over time. Novel experiences in mice lead to opposing effects on inhibition of Fos -activated hippocampal CA1 pyramidal neurons by parvalbumin- and cholecystokinin-expressing interneurons, revealing the roles of FOS and SCG2 in neural plasticity and consolidation of memories.

Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons 1 – 5 . Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4–TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4–NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4–NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4–NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing. A neuron-specific activity-dependent DNA repair mechanism is identified, the impairment of which may lead to neurodevelopmental disorders, neurodegeneration and ageing.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.

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.

Large herbivores are capable of modifying entire ecosystems with a combination of direct (for example browsing/grazing, trampling, defecation) and indirect (for example affecting plant species composition that then alters soil properties) effects. With many ungulate populations increasing across the northern hemisphere it is important to develop a general theory for how these animals can be expected to impact their habitats. Here we present the results of an 8-year experimental exclusion of moose (Alces alces) from 15 recent boreal forest clear-cut sites in Central Norway. We used standard univariate techniques to describe the treatment effect on multiple forest and soil properties and combined this with a multivariate Bayesian network structure learning approach to objectively assess the potential mechanistic pathways for indirect effects on soils and soil fertility. We found that excluding moose had predictable direct effects, such as increasing the ratio of deciduous to coniferous tree biomass and the canopy cover and decreasing soil bulk density and temperature. However, we found no treatment effects on any measures of soil processes or quality (decomposition, nitrogen availability, C/N ratio, pH, nutrient stocks), and furthermore, we found only limited evidence that the direct effects had cascading (indirect) effects on soils. These findings oppose the commonly held belief that moose exclusion will increase soil fertility, but still highlights the strong ability of moose to directly modify forested ecosystems.

Mutations in the GJB2 gene cause DFNB1, the most common hereditary hearing loss. GJB2 is expressed by cochlear epithelial cells and fibrocytes, but not by sensory hair cells or neurons. Attempts to treat DFNB1 mouse models with gene therapy have not substantially restored function, as inappropriate expression in hair cells and neurons might compromise their electrical activity. Here, we use ATAC-seq to identify candidate gene regulatory elements (GREs) that can drive cell-type-specific expression of GJB2 . HA-tagged GJB2, delivered to a conditional knockout mouse with AAV vectors carrying GREs, is expressed by the appropriate cells, prevents degeneration, and rescues hearing by only 10–20 dB. In a Gjb2 partial knockdown model, a vector lacking HA prevents degeneration and completely restores hearing. In cynomolgus monkey cochleas, human GJB2.HA delivered with similar vectors is located in the appropriate cell types and causes little or no compromise of hearing sensitivity. Together, these findings suggest that GRE-mediated expression of GJB2 can prevent hearing loss in DFNB1 patients. Mutations in GJB2 cause DFNB1, the most common hereditary deafness. ATAC-seq identification of gene regulatory elements enables targeted GJB2 delivery to cochlear cells, preventing hearing loss in mouse models.

The septum is a ventral forebrain structure known to regulate innate behaviors. During embryonic development, septal neurons are produced in multiple proliferative areas from neural progenitors following transcriptional programs that are still largely unknown. Here, we use a combination of single-cell RNA sequencing, histology, and genetic models to address how septal neuron diversity is established during neurogenesis. We find that the transcriptional profiles of septal progenitors change along neurogenesis, coinciding with the generation of distinct neuron types. We characterize the septal eminence, an anatomically distinct and transient proliferative zone composed of progenitors with distinctive molecular profiles, proliferative capacity, and fate potential compared to the rostral septal progenitor zone. We show that Nkx2.1 -expressing septal eminence progenitors give rise to neurons belonging to at least three morphological classes, born in temporal cohorts that are distributed across different septal nuclei in a sequential fountain-like pattern. Our study provides insight into the molecular programs that control the sequential production of different neuronal types in the septum, a structure with important roles in regulating mood and motivation.