A chromosome region linked to neurodevelopmental disorders acts in distinct neuronal circuits in males and females to control locomotor behavior.

Biological sex shapes the manifestation and progression of neurodevelopmental disorders (NDDs). These disorders often demonstrate male-specific vulnerabilities; however, the identification of underlying mechanisms remains a significant challenge in the field. Hemideletion of the 16p11.2 region (16p11.2 del/+) is associated with NDDs, and mice modeling 16p11.2 del/+ exhibit sex-specific striatum-related phenotypes relevant to NDDs. Striatal circuits, crucial for locomotor control, consist of two distinct pathways: the direct and indirect pathways originating from D1 dopamine receptor (D1R) and D2 dopamine receptor (D2R) expressing spiny projection neurons (SPNs), respectively. In this study, we define the impact of 16p11.2 del/+ on striatal circuits in male and female mice. Using snRNA-seq, we identify sex- and cell type-specific transcriptomic changes in the D1- and D2-SPNs of 16p11.2 del/+ mice, indicating distinct transcriptomic signatures in D1-SPNs and D2-SPNs in males and females, with a ∼5-fold greater impact in males. Further pathway analysis reveals differential gene expression changes in 16p11.2 del/+ male mice linked to synaptic plasticity in D1- and D2-SPNs and GABA signaling pathway changes in D1-SPNs. Consistent with our snRNA-seq study revealing changes in GABA signaling pathways, we observe distinct changes in miniature inhibitory postsynaptic currents (mIPSCs) in D1- and D2-SPNs from 16p11.2 del/+ male mice. Behaviorally, we utilize conditional genetic approaches to introduce the hemideletion selectively in either D1- or D2-SPNs and find that conditional hemideletion of genes in the 16p11.2 region in D2-SPNs causes hyperactivity in male mice, but hemideletion in D1-SPNs does not. Within the striatum, hemideletion of genes in D2-SPNs in the dorsal lateral striatum leads to hyperactivity in males, demonstrating the importance of this striatal region. Interestingly, conditional 16p11.2 del/+ within the cortex drives hyperactivity in both sexes. Our work reveals that a locus linked to NDDs acts in different striatal circuits, selectively impacting behavior in a sex- and cell type-specific manner, providing new insight into male vulnerability for NDDs. Highlights: - 16p11.2 hemideletion (16p11.2 del/+) induces sex- and cell type-specific transcriptomic signatures in spiny projection neurons (SPNs). - Transcriptomic changes in GABA signaling in D1-SPNs align with changes in inhibitory synapse function. - 16p11.2 del/+ in D2-SPNs causes hyperactivity in males but not females. - 16p11.2 del/+ in D2-SPNs in the dorsal lateral striatum drives hyperactivity in males. - 16p11.2 del/+ in cortex drives hyperactivity in both sexes.

Using deep learning to quantify neuronal activation from single-cell and spatial transcriptomic data.

Neuronal activity-dependent transcription directs molecular processes that regulate synaptic plasticity, brain circuit development, behavioral adaptation, and long-term memory. Single cell RNA-sequencing technologies (scRNAseq) are rapidly developing and allow for the interrogation of activity-dependent transcription at cellular resolution. Here, we present NEUROeSTIMator, a deep learning model that integrates transcriptomic signals to estimate neuronal activation in a way that we demonstrate is associated with Patch-seq electrophysiological features and that is robust against differences in species, cell type, and brain region. We demonstrate this method's ability to accurately detect neuronal activity in previously published studies of single cell activity-induced gene expression. Further, we applied our model in a spatial transcriptomic study to identify unique patterns of learning-induced activity across different brain regions in male mice. Altogether, our findings establish NEUROeSTIMator as a powerful and broadly applicable tool for measuring neuronal activation, whether as a critical covariate or a primary readout of interest.

Dissecting 16p11.2 hemi-deletion to study sex-specific striatal phenotypes of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are polygenic in nature and copy number variants (CNVs) are ideal candidates to study the nature of this polygenic risk. The disruption of striatal circuits is considered a central mechanism in NDDs. The 16p11.2 hemi-deletion (16p11.2 del/+) is one of the most common CNVs associated with NDD, and 16p11.2 del/+ mice show sex-specific striatum-related behavioral phenotypes. However, the critical genes among the 27 genes in the 16p11.2 region that underlie these phenotypes remain unknown. Previously, we applied a novel strategy to identify candidate genes associated with the sex-specific phenotypes of 16p11.2 del/+ mice and highlighted three genes within the deleted region: thousand and one amino acid protein kinase 2 (Taok2), seizure-related 6 homolog-like 2 (Sez6l2), and major vault protein (Mvp). Using CRISPR/Cas9, we generated mice carrying null mutations in Taok2, Sez6l2, and Mvp (3 gene hemi-deletion (3g del/+)). Hemi-deletion of these 3 genes recapitulates sex-specific behavioral alterations in striatum-dependent behavioral tasks observed in 16p11.2 del/+ mice, specifically male-specific hyperactivity and impaired motivation for reward seeking. Moreover, RNAseq analysis revealed that 3g del/+ mice exhibit gene expression changes in the striatum similar to 16p11.2 del/+ mice exclusively in males. Subsequent analysis identified translation dysregulation and/or extracellular signal-regulated kinase signaling as plausible molecular mechanisms underlying male-specific, striatum-dependent behavioral alterations. Interestingly, ribosomal profiling supported the notion of translation dysregulation in both 3g del/+ and 16p11.2 del/+ male mice. However, mice carrying a 4-gene deletion (with an additional deletion of Mapk3) exhibited fewer phenotypic similarities with 16p11.2 del/+ mice. Together, the mutation of 3 genes within the 16p11.2 region phenocopies striatal sex-specific phenotypes of 16p11.2 del/+ mice. These results support the importance of a polygenic approach to study NDDs and underscore that the effects of the large genetic deletions result from complex interactions between multiple candidate genes.

Spatial transcriptomics reveals unique gene expression changes in different brain regions after sleep deprivation.

Sleep deprivation has far-reaching consequences on the brain and behavior, impacting memory, attention, and metabolism. Previous research has focused on gene expression changes in individual brain regions, such as the hippocampus or cortex. Therefore, it is unclear how uniformly or heterogeneously sleep loss affects the brain. Here, we use spatial transcriptomics to define the impact of a brief period of sleep deprivation across the brain in male mice. We find that sleep deprivation induced pronounced differences in gene expression across the brain, with the greatest changes in the hippocampus, neocortex, hypothalamus, and thalamus. Both the differentially expressed genes and the direction of regulation differed markedly across regions. Importantly, we developed bioinformatic tools to register tissue sections and gene expression data into a common anatomical space, allowing a brain-wide comparison of gene expression patterns between samples. Our results suggest that distinct molecular mechanisms acting in discrete brain regions underlie the biological effects of sleep deprivation.

Activity-induced gene expression in the human brain.

Activity-induced gene expression underlies synaptic plasticity and brain function. Here, using molecular sequencing techniques, we define activity-dependent transcriptomic and epigenomic changes at the tissue and single-cell level in the human brain following direct electrical stimulation of the anterior temporal lobe in patients undergoing neurosurgery. Genes related to transcriptional regulation and microglia-specific cytokine activity displayed the greatest induction pattern, revealing a precise molecular signature of neuronal activation in the human brain.

Mapping the spatial transcriptomic signature of the hippocampus during memory consolidation.

Memory consolidation involves discrete patterns of transcriptional events in the hippocampus. Despite the emergence of single-cell transcriptomic profiling techniques, mapping the transcriptomic signature across subregions of the hippocampus has remained challenging. Here, we utilized unbiased spatial sequencing to delineate transcriptome-wide gene expression changes across subregions of the dorsal hippocampus of male mice following learning. We find that each subregion of the hippocampus exhibits distinct yet overlapping transcriptomic signatures. The CA1 region exhibited increased expression of genes related to transcriptional regulation, while the DG showed upregulation of genes associated with protein folding. Importantly, our approach enabled us to define the transcriptomic signature of learning within two less-defined hippocampal subregions, CA1 stratum radiatum, and oriens. We demonstrated that CA1 subregion-specific expression of a transcription factor subfamily has a critical functional role in the consolidation of long-term memory. This work demonstrates the power of spatial molecular approaches to reveal simultaneous transcriptional events across the hippocampus during memory consolidation.

Dissecting 16p11.2 hemi-deletion to study sex-specific striatal phenotypes of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are polygenic in nature and copy number variants (CNVs) are ideal candidates to study the nature of this polygenic risk. The disruption of striatal circuits is considered a central mechanism in NDDs. The 16p11.2 hemi-deletion (16p11.2 del) is one of the most common CNVs associated with NDD, and 16p11.2 del/+ mice show sex-specific striatum-related behavioral phenotypes. However, the critical genes among the 27 genes in the 16p11.2 region that underlie these phenotypes remain unknown. Previously, we applied a novel strategy to identify candidate genes associated with the sex-specific phenotypes of 16p11.2 del/+ mice and identified 3 genes of particular importance within the deleted region: thousand and one amino acid protein kinase 2 (Taok2), seizure-related 6 homolog-like 2 (Sez6l2), and major vault protein (Mvp). Using the CRISPR/Cas9 technique, we generated 3 gene hemi-deletion (3g del/+) mice carrying null mutations in Taok2, Sez6l2, and Mvp. We assessed striatum-dependent phenotypes of these 3g del/+ mice in behavioral, molecular, and imaging studies. Hemi-deletion of Taok2, Sez6l2, and Mvp induces sex-specific behavioral alterations in striatum-dependent behavioral tasks, specifically male-specific hyperactivity and impaired motivation for reward seeking, resembling behavioral phenotypes of 16p11.2 del/+ mice. Moreover, RNAseq analysis revealed that 3g del/+ mice exhibit gene expression changes in the striatum similar to 16p11.2 del/+ mice, but only in males. Pathway analysis identified ribosomal dysfunction and translation dysregulation as molecular mechanisms underlying male-specific, striatum-dependent behavioral alterations. Together, the mutation of 3 genes within the 16p11.2 region phenocopies striatal sex-specific phenotypes of 16p11.2 del/+ mice, unlike single gene mutation studies. These results support the importance of a polygenic approach to study NDDs and our novel strategy to identify genes of interest using gene expression patterns in brain regions, such as the striatum, which are impacted in these disorders.

Dissecting 16p11.2 hemi-deletion to study sex-specific striatal phenotypes of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are polygenic in nature and copy number variants (CNVs) are ideal candidates to study the nature of this polygenic risk. The disruption of striatal circuits is considered a central mechanism in NDDs. The 16p11.2 hemi-deletion (16p11.2 del) is one of the most common CNVs associated with NDD, and 16p11.2 del/+ mice show sex-specific striatum-related behavioral phenotypes. However, the critical genes among the 27 genes in the 16p11.2 region that underlie these phenotypes remain unknown. Previously, we applied a novel strategy to identify candidate genes associated with the sex-specific phenotypes of 16p11.2 del/+ mice and identified 3 genes of particular importance within the deleted region: thousand and one amino acid protein kinase 2 ( Taok2 ), seizure-related 6 homolog-like 2 ( Sez6l2 ), and major vault protein ( Mvp ). Using the CRISPR/Cas9 technique, we generated 3 gene hemi-deletion (3g del/+) mice carrying null mutations in Taok2, Sez6l2 , and Mvp . We assessed striatum-dependent phenotypes of these 3g del/+ mice in behavioral, molecular, and imaging studies. Hemi-deletion of Taok2, Sez6l2 , and Mvp induces sex-specific behavioral alterations in striatum-dependent behavioral tasks, specifically male-specific hyperactivity and impaired motivation for reward seeking, resembling behavioral phenotypes of 16p11.2 del/+ mice. Moreover, RNAseq analysis revealed that 3g del/+ mice exhibit gene expression changes in the striatum similar to 16p11.2 del/+ mice, but only in males. Pathway analysis identified ribosomal dysfunction and translation dysregulation as molecular mechanisms underlying male-specific, striatum-dependent behavioral alterations. Together, the mutation of 3 genes within the 16p11.2 region phenocopies striatal sex-specific phenotypes of 16p11.2 del/+ mice, unlike single gene mutation studies. These results support the importance of a polygenic approach to study NDDs and our novel strategy to identify genes of interest using gene expression patterns in brain regions, such as the striatum, which are impacted in these disorders.

Sleep and memory: The impact of sleep deprivation on transcription, translational control, and protein synthesis in the brain.

In countries around the world, sleep deprivation represents a widespread problem affecting school-age children, teenagers, and adults. Acute sleep deprivation and more chronic sleep restriction adversely affect individual health, impairing memory and cognitive performance as well as increasing the risk and progression of numerous diseases. In mammals, the hippocampus and hippocampus-dependent memory are vulnerable to the effects of acute sleep deprivation. Sleep deprivation induces changes in molecular signaling, gene expression and may cause changes in dendritic structure in neurons. Genome wide studies have shown that acute sleep deprivation alters gene transcription, although the pool of genes affected varies between brain regions. More recently, advances in research have drawn attention to differences in gene regulation between the level of the transcriptome compared with the pool of mRNA associated with ribosomes for protein translation following sleep deprivation. Thus, in addition to transcriptional changes, sleep deprivation also affects downstream processes to alter protein translation. In this review, we focus on the multiple levels through which acute sleep deprivation impacts gene regulation, highlighting potential post-transcriptional and translational processes that may be affected by sleep deprivation. Understanding the multiple levels of gene regulation impacted by sleep deprivation is essential for future development of therapeutics that may mitigate the effects of sleep loss.

Spatial transcriptomics reveals unique gene expression changes in different brain regions after sleep deprivation.

Sleep deprivation has far-reaching consequences on the brain and behavior, impacting memory, attention, and metabolism. Previous research has focused on gene expression changes in individual brain regions, such as the hippocampus or cortex. Therefore, it is unclear how uniformly or heterogeneously sleep loss affects the brain. Here, we use spatial transcriptomics to define the impact of a brief period of sleep deprivation across the brain. We find that sleep deprivation induced pronounced differences in gene expression across the brain, with the greatest changes in the hippocampus, neocortex, hypothalamus, and thalamus. Both the differentially expressed genes and the direction of regulation differed markedly across regions. Importantly, we developed bioinformatic tools to register tissue sections and gene expression data into a common anatomical space, allowing a brain-wide comparison of gene expression patterns between samples. Our results suggest that distinct molecular mechanisms acting in discrete brain regions underlie the biological effects of sleep deprivation.

Endoplasmic reticulum chaperone genes encode effectors of long-term memory.

The mechanisms underlying memory loss associated with Alzheimer's disease and related dementias (ADRD) remain unclear, and no effective treatments exist. Fundamental studies have shown that a set of transcriptional regulatory proteins of the nuclear receptor 4a (Nr4a) family serve as molecular switches for long-term memory. Here, we show that Nr4a proteins regulate the transcription of genes encoding chaperones that localize to the endoplasmic reticulum (ER). These chaperones fold and traffic plasticity-related proteins to the cell surface during long-lasting forms of synaptic plasticity and memory. Dysregulation of Nr4a transcription factors and ER chaperones is linked to ADRD, and overexpressing Nr4a1 or the chaperone Hspa5 ameliorates long-term memory deficits in a tau-based mouse model of ADRD, pointing toward innovative therapeutic approaches for treating memory loss. Our findings establish a unique molecular concept underlying long-term memory and provide insights into the mechanistic basis of cognitive deficits in dementia.

Translational changes induced by acute sleep deprivation uncovered by TRAP-Seq.

Sleep deprivation is a global health problem adversely affecting health as well as causing decrements in learning and performance. Sleep deprivation induces significant changes in gene transcription in many brain regions, with the hippocampus particularly susceptible to acute sleep deprivation. However, less is known about the impacts of sleep deprivation on post-transcriptional gene regulation. To identify the effects of sleep deprivation on the translatome, we took advantage of the RiboTag mouse line to express HA-labeled Rpl22 in CaMKIIα neurons to selectively isolate and sequence mRNA transcripts associated with ribosomes in excitatory neurons. We found 198 differentially expressed genes in the ribosome-associated mRNA subset after sleep deprivation. In comparison with previously published data on gene expression in the hippocampus after sleep deprivation, we found that the subset of genes affected by sleep deprivation was considerably different in the translatome compared with the transcriptome, with only 49 genes regulated similarly. Interestingly, we found 478 genes differentially regulated by sleep deprivation in the transcriptome that were not significantly regulated in the translatome of excitatory neurons. Conversely, there were 149 genes differentially regulated by sleep deprivation in the translatome but not in the whole transcriptome. Pathway analysis revealed differences in the biological functions of genes exclusively regulated in the transcriptome or translatome, with protein deacetylase activity and small GTPase binding regulated in the transcriptome and unfolded protein binding, kinase inhibitor activity, neurotransmitter receptors and circadian rhythms regulated in the translatome. These results indicate that sleep deprivation induces significant changes affecting the pool of actively translated mRNAs.

Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

The Transferable Resistome of Produce.

Produce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistant Escherichia coli were isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistant E. coli isolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. TET-resistant E. coli isolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids and blaCTX-M-1 From mixed salad and cilantro, IncF, IncI1, and IncP-1ß plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing into E. coli suggests that they could transfer to gut bacteria as well.IMPORTANCE Produce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI plasmids were the most prevalent plasmid types in E. coli isolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.