Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons.

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation system in which catalytically dead Cas9 fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs, resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

SUFI: an automated approach to spectral unmixing of fluorescent multiplex images captured in mouse and post-mortem human brain tissues.

BACKGROUND: Multispectral fluorescence imaging coupled with linear unmixing is a form of image data collection and analysis that allows for measuring multiple molecular signals in a single biological sample. Multiple fluorescent dyes, each measuring a unique molecule, are simultaneously measured and subsequently "unmixed" to provide a read-out for each molecular signal. This strategy allows for measuring highly multiplexed signals in a single data capture session, such as multiple proteins or RNAs in tissue slices or cultured cells, but can often result in mixed signals and bleed-through problems across dyes. Existing spectral unmixing algorithms are not optimized for challenging biological specimens such as post-mortem human brain tissue, and often require manual intervention to extract spectral signatures. We therefore developed an intuitive, automated, and flexible package called SUFI: spectral unmixing of fluorescent images. RESULTS: This package unmixes multispectral fluorescence images by automating the extraction of spectral signatures using vertex component analysis, and then performs one of three unmixing algorithms derived from remote sensing. We evaluate these remote sensing algorithms' performances on four unique biological datasets and compare the results to unmixing results obtained using ZEN Black software (Zeiss). We lastly integrate our unmixing pipeline into the computational tool dotdotdot, which is used to quantify individual RNA transcripts at single cell resolution in intact tissues and perform differential expression analysis, and thereby provide an end-to-end solution for multispectral fluorescence image analysis and quantification. CONCLUSIONS: In summary, we provide a robust, automated pipeline to assist biologists with improved spectral unmixing of multispectral fluorescence images.

Proteasome modulates positive and negative translational regulators in long-term synaptic plasticity.

Proteolysis by the ubiquitin-proteasome pathway appears to have a complex role in synaptic plasticity, but its various functions remain to be elucidated. Using late phase long-term potentiation (L-LTP) in the hippocampus of the mouse as a model for long-term synaptic plasticity, we previously showed that inhibition of the proteasome enhances induction but blocks maintenance of L-LTP. In this study, we investigated the possible mechanisms by which proteasome inhibition has opposite effects on L-LTP induction and maintenance. Our results show that inhibiting phosphatidyl inositol-3 kinase or blocking the interaction between eukaryotic initiation factors 4E (eIF4E) and 4G (eIF4G) reduces the enhancement of L-LTP induction brought about by proteasome inhibition suggesting interplay between proteolysis and the signaling pathway mediated by mammalian target of rapamycin (mTOR). Also, proteasome inhibition leads to accumulation of translational activators in the mTOR pathway such as eIF4E and eukaryotic elongation factor 1A (eEF1A) early during L-LTP causing increased induction. Furthermore, inhibition of the proteasome causes a buildup of translational repressors, such as polyadenylate-binding protein interacting protein 2 (Paip2) and eukaryotic initiation factor 4E-binding protein 2 (4E-BP2), during late stages of L-LTP contributing to the blockade of L-LTP maintenance. Thus, the proteasome plays a critical role in regulating protein synthesis during L-LTP by tightly controlling translation. Our results provide novel mechanistic insights into the interplay between protein degradation and protein synthesis in long-term synaptic plasticity.

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Background: Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. Results: We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. Conclusions: visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStiched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStiched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Transcriptomic characterization of human lateral septum neurons reveals conserved and divergent marker genes across species.

The lateral septum (LS) is a midline, subcortical structure, which regulates social behaviors that are frequently impaired in neurodevelopmental disorders including schizophrenia and autism spectrum disorder. Mouse studies have identified neuronal populations within the LS that express a variety of molecular markers, including vasopressin receptor, oxytocin receptor, and corticotropin releasing hormone receptor, which control specific facets of social behavior. Despite its critical role in regulating social behavior and notable gene expression patterns, comprehensive molecular profiling of the human LS has not been performed. Here, we conducted single nucleus RNA-sequencing (snRNA-seq) to generate the first transcriptomic profiles of the human LS using postmortem human brain tissue samples from 3 neurotypical donors. Our analysis identified 5 transcriptionally distinct neuronal cell types within the human LS that are enriched for TRPC4, the gene encoding Trp-related protein 4. Differential expression analysis revealed a distinct LS neuronal cell type that is enriched for OPRM1, the gene encoding the µ-opioid receptor. Leveraging recently generated mouse LS snRNA-seq datasets, we conducted a cross-species analysis. Our results demonstrate that TRPC4 enrichment in the LS is highly conserved between human and mouse, while FREM2, which encodes FRAS1 related extracellular matrix protein 2, is enriched only in the human LS. Together, these results highlight transcriptional heterogeneity of the human LS, and identify robust marker genes for the human LS.

Transcriptomic diversity of amygdalar subdivisions across humans and nonhuman primates.

The amygdaloid complex mediates learning, memory, and emotions. Understanding the cellular and anatomical features that are specialized in the amygdala of primates versus other vertebrates requires a systematic, anatomically-resolved molecular analysis of constituent cell populations. We analyzed five nuclear subdivisions of the primate amygdala with single-nucleus RNA sequencing in macaques, baboons, and humans to examine gene expression profiles for excitatory and inhibitory neurons and confirmed our results with single-molecule FISH analysis. We identified distinct subtypes of FOXP2 + interneurons in the intercalated cell masses and protein-kinase C-δ interneurons in the central nucleus. We also establish that glutamatergic, pyramidal-like neurons are transcriptionally specialized within the basal, lateral, or accessory basal nuclei. Understanding the molecular heterogeneity of anatomically-resolved amygdalar neuron types provides a cellular framework for improving existing models of how amygdalar neural circuits contribute to cognition and mental health in humans by using nonhuman primates as a translational bridge.

Transcriptomic analysis of the human habenula in schizophrenia.

Pathophysiology of many neuropsychiatric disorders, including schizophrenia (SCZD), is linked to habenula (Hb) function. While pharmacotherapies and deep brain stimulation targeting the Hb are emerging as promising therapeutic treatments, little is known about the cell type-specific transcriptomic organization of the human Hb or how it is altered in SCZD. Here we define the molecular neuroanatomy of the human Hb and identify transcriptomic changes in individuals with SCZD compared to neurotypical controls. Utilizing Hb-enriched postmortem human brain tissue, we performed single nucleus RNA-sequencing (snRNA-seq; n=7 neurotypical donors) and identified 17 molecularly defined Hb cell types across 16,437 nuclei, including 3 medial and 7 lateral Hb populations, several of which were conserved between rodents and humans. Single molecule fluorescent in situ hybridization (smFISH; n=3 neurotypical donors) validated snRNA-seq Hb cell types and mapped their spatial locations. Bulk RNA-sequencing and cell type deconvolution in Hb-enriched tissue from 35 individuals with SCZD and 33 neurotypical controls yielded 45 SCZD-associated differentially expressed genes (DEGs, FDR < 0.05), with 32 (71%) unique to Hb-enriched tissue. eQTL analysis identified 717 independent SNP-gene pairs (FDR < 0.05), where either the SNP is a SCZD risk variant (16 pairs) or the gene is a SCZD DEG (7 pairs). eQTL and SCZD risk colocalization analysis identified 16 colocalized genes. These results identify topographically organized cell types with distinct molecular signatures in the human Hb and demonstrate unique genetic changes associated with SCZD, thereby providing novel molecular insights into the role of Hb in neuropsychiatric disorders. One Sentence Summary: Transcriptomic analysis of the human habenula and identification of molecular changes associated with schizophrenia risk and illness state.

An integrated single-nucleus and spatial transcriptomics atlas reveals the molecular landscape of the human hippocampus.

The hippocampus contains many unique cell types, which serve the structure's specialized functions, including learning, memory and cognition. These cells have distinct spatial topography, morphology, physiology, and connectivity, highlighting the need for transcriptome-wide profiling strategies that retain cytoarchitectural organization. Here, we generated spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq) data from adjacent tissue sections of the anterior human hippocampus across ten adult neurotypical donors. We defined molecular profiles for hippocampal cell types and spatial domains. Using non-negative matrix factorization and transfer learning, we integrated these data to define gene expression patterns within the snRNA-seq data and infer the expression of these patterns in the SRT data. With this approach, we leveraged existing rodent datasets that feature information on circuit connectivity and neural activity induction to make predictions about axonal projection targets and likelihood of ensemble recruitment in spatially-defined cellular populations of the human hippocampus. Finally, we integrated genome-wide association studies with transcriptomic data to identify enrichment of genetic components for neurodevelopmental, neuropsychiatric, and neurodegenerative disorders across cell types, spatial domains, and gene expression patterns of the human hippocampus. To make this comprehensive molecular atlas accessible to the scientific community, both raw and processed data are freely available, including through interactive web applications.

Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons.

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation (CRISPRa) system in which catalytically-dead Cas9 (dCas9) fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs (sgRNAs), resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

Pharmacologic inhibition of LIMK1 provides dendritic spine resilience against ß-amyloid.

Alzheimer's disease (AD) therapies predominantly focus on ß-amyloid (Aß), but Aß effects may be maximal before clinical symptoms appear. Downstream of Aß, dendritic spine loss correlates most strongly with cognitive decline in AD. Rho-associated kinases (ROCK1 and ROCK2) regulate the actin cytoskeleton, and ROCK1 and ROCK2 protein abundances are increased in early AD. Here, we found that the increased abundance of ROCK1 in cultured primary rat hippocampal neurons reduced dendritic spine length through a myosin-based pathway, whereas the increased abundance of ROCK2 induced spine loss through the serine and threonine kinase LIMK1. Aß42 oligomers can activate ROCKs. Here, using static imaging studies combined with multielectrode array analyses, we found that the ROCK2-LIMK1 pathway mediated Aß42-induced spine degeneration and neuronal hyperexcitability. Live-cell microscopy revealed that pharmacologic inhibition of LIMK1 rendered dendritic spines resilient to Aß42 oligomers. Treatment of hAPP mice with a LIMK1 inhibitor rescued Aß-induced hippocampal spine loss and morphologic aberrations. Our data suggest that therapeutically targeting LIMK1 may provide dendritic spine resilience to Aß and therefore may benefit cognitively normal patients that are at high risk for developing dementia.

A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

Proteasome limits plasticity-related signaling to the nucleus in the hippocampus.

Proteolysis by the ubiquitin-proteasome pathway has pleiotropic effects on both induction and maintenance of long-term synaptic plasticity. In this study, we examined the effect of proteasome inhibition on signaling to the nucleus during late-phase long-term potentiation. When a subthreshold L-LTP induction protocol was used, proteasome inhibition led to a significant increase in phosphorylated CREB (pCREB) in the nucleus. Inhibitors of cAMP-dependent protein kinase/protein kinase A, extracellular signal-regulated kinase and cGMP-dependent protein kinase/protein kinase G all blocked the proteasome-inhibition-mediated increase in nuclear pCREB after subthreshold stimulation. These results lay the groundwork for understanding a novel role for the proteasome in limiting signaling to the nucleus in the absence of adequate synaptic stimulation.

The proteasome and epigenetics: zooming in on histone modifications.

The proteasome is a structural complex of many proteins that degrades substrates marked by covalent linkage to ubiquitin. Many years of research has shown a role for ubiquitin-proteasome-mediated proteolysis in synaptic plasticity and memory mainly in degrading synaptic, cytoplasmic and nuclear proteins. Recent work indicates that the proteasome has wider proteolytic and non-proteolytic roles in processes such as histone modifications that affect synaptic plasticity and memory. In this review, we assess the evidence gathered from neuronal as well as non-neuronal cell types regarding the function of the proteasome in positive or negative regulation of posttranslational modifications of histones, such as acetylation, methylation and ubiquitination. We discuss the critical roles of the proteasome in clearing excess histone proteins in various cellular contexts and the possible non-proteolytic functions in regulating transcription of target genes. In addition, we summarize the current literature on diverse chromatin-remodeling machineries, such as histone acetyltransferases, deacetylates, methyltransferases and demethylases, as targets for proteasomal degradation across experimental models. Lastly, we provide a perspective on how proteasomal regulation of histone modifications may modulate synaptic plasticity in the nervous system.

Proteasome regulates transcription-favoring histone methylation, acetylation and ubiquitination in long-term synaptic plasticity.

Histone modifications, such as lysine methylation, acetylation and ubiquitination, are epigenetic tags that shape the chromatin landscape and regulate transcription required for synaptic plasticity and memory. Here, we show that transcription-promoting histone H3 trimethylated at lysine 4 (H3K4me3), histone H3 acetylated at lysine 9 and 14 (H3K9/14ac), and histone H2B monoubiquitinated at lysine 120 (H2BK120ub) are enhanced after the induction of long-lasting chemically-induced long-term potentiation (cLTP) in the murine hippocampus. While H3K4me3 and H3K9/14ac were transiently upregulated, H2BK120ub levels oscillated after cLTP induction. In addition, we present results showing that blocking the proteasome, a molecular complex specialized for targeted protein degradation, inhibited the upregulation of these epigenetic tags after cLTP. Thus, our study provides the initial steps toward understanding the role of the proteasome in regulating histone modifications critical for synaptic plasticity.

Local ubiquitin-proteasome-mediated proteolysis and long-term synaptic plasticity.

The ubiquitin-proteasome pathway (UPP) of protein degradation has many roles in synaptic plasticity that underlies memory. Work on both invertebrate and vertebrate model systems has shown that the UPP regulates numerous substrates critical for synaptic plasticity. Initial research took a global view of ubiquitin-protein degradation in neurons. Subsequently, the idea of local protein degradation was proposed a decade ago. In this review, we focus on the functions of the UPP in long-term synaptic plasticity and discuss the accumulated evidence in support of the idea that the components of the UPP often have disparate local roles in different neuronal compartments rather than a single cell-wide function.