A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

Dynamics of reactive astrocytes fosters tissue regeneration after cuprizone-induced demyelination.

Demyelination in the central nervous system (CNS) is a hallmark of many neurodegenerative diseases such as multiple sclerosis (MS) and others. Here, we studied astrocytes during de- and remyelination in the cuprizone mouse model. To this end, we exploited the ribosomal tagging (RiboTag) technology that is based on Cre-mediated cell type-selective HA-tagging of ribosomes. Analyses were performed in the corpus callosum of GFAP-Cre+/- Rpl22HA/wt mice 5 weeks after cuprizone feeding, at the peak of demyelination, and 0.5 and 2 weeks after cuprizone withdrawal, when remyelination and tissue repair is initiated. After 5 weeks of cuprizone feeding, reactive astrocytes showed inflammatory signatures with enhanced expression of genes that modulate leukocyte migration (Tlr2, Cd86, Parp14) and they produced the chemokine CXCL10, as verified by histology. Furthermore, demyelination-induced reactive astrocytes expressed numerous ligands including Cx3cl1, Csf1, Il34, and Gas6 that act on homeostatic as well as activated microglia and thus potentially mediate activation and recruitment of microglia and enhancement of their phagocytotic activity. During early remyelination, HA-tagged cells displayed reduced inflammatory response signatures, as indicated by shutdown of CXCL10 production, and enhanced expression of osteopontin (SPP1) as well as of factors that are relevant for tissue remodeling (Timp1), regeneration and axonal repair. During late remyelination, the signatures shifted towards resolving inflammation by active suppression of lymphocyte activation and differentiation and support of glia cell differentiation. In conclusion, we detected highly dynamic astroglial transcriptomic signatures in the cuprizone model, which reflects excessive communication among glia cells and highlights different astrocyte functions during neurodegeneration and regeneration.

Cell type- and region-specific translatomes in an MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by the progressive loss of nigrostriatal dopaminergic neurons (DANs), involving the dysregulation of both neurons and glial cells. Cell type- and region-specific gene expression profiles can provide an effective source for revealing the mechanisms of PD. In this study, we adopted the RiboTag approach to obtain cell type (DAN, microglia, astrocytes)- and brain region (substantia nigra, caudate-putamen)-specific translatomes at an early stage in an MPTP-induced mouse model of PD. Through DAN-specific translatome analysis, the glycosphingolipid biosynthetic process was identified as a significantly downregulated pathway in the MPTP-treated mice. ST8Sia6, a key downregulated gene related to glycosphingolipid biosynthesis, was confirmed to be downregulated in nigral DANs from postmortem brains of patients with PD. Specific expression of ST8Sia6 in DANs exerts anti-inflammatory and neuroprotective effects in MPTP-treated mice. Through cell type (microglia vs. astrocyte) and brain region (substantia nigra vs. caudate-putamen) comparisons, nigral microglia showed the most intense immune responses. Microglia and astrocytes in the substantia nigra showed similar levels of activation in interferon-related pathways and interferon gamma (IFNG) was identified as the top upstream regulator in both cell types. This work highlights that the glycosphingolipid metabolism pathway in the DAN is involved in neuroinflammation and neurodegeneration in an MPTP mouse model of PD and provides a new data source for elucidating the pathogenesis of PD.

Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

ß-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery.

BACKGROUND: Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS: Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of ß-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS: With ~ 4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of ß-adrenergic downstream signaling triggered enteric glial reactivity. Finally, distinct adrenergic agonists revealed ß-1/2 adrenoceptors as the molecular targets of sympathetic-driven enteric glial reactivity. CONCLUSIONS: Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active ß-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

A conserved pathway of transdifferentiation in murine Kupffer cells.

Abundant long-lived liver-resident macrophages, termed Kupffer cells, are activated during chronic liver injury. They secrete both pro-inflammatory and pro-fibrotic cytokines, which act on hepatic stellate cells causing their transdifferentiation into myofibroblasts that deposit collagen. In other tissues, wound-associated macrophages go further, and transdifferentiate into fibrocytes, secreting collagen themselves. We tested Kupffer cells for this property in two experimental models: mixed non-parenchymal cell culture, and precision-cut liver slice culture. Using the Emr1-Cre transgene as a driver and the RiboTag transgene as a reporter, we found that Kupffer cells undergo transdifferentiation under these circumstances. Over time, they lose the expression of both Kupffer cell-specific and macrophage-specific genes and the transcription factors that control their expression, and they begin to express multiple genes and proteins characteristic of either myofibroblasts or tissue fibroblasts. These effects were strongly conserved between non-parenchymal cell culture and liver tissue slice culture, arguing that such transdifferentiation is a conserved function of Kupffer cells. We conclude that in addition to supporting fibrosis through an action on stellate cells, Kupffer cells also participate in liver fibrosis through transdifferentiation into fibrocytes.

Expression of the regulated isoform of the electrogenic Na+/HCO3- cotransporter, NBCe1, is enriched in pacemaker interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

High Sensitivity Mapping of Cortical Dopamine D2 Receptor Expressing Neurons.

Cortical D2 dopamine receptor (Drd2) have mostly been examined in the context of cognitive function regulation and neurotransmission modulation of medial prefrontal cortex by principal neurons and parvalbumin positive, fast-spiking, interneurons in schizophrenia. Early studies suggested the presence of D2 receptors in several cortical areas, albeit with major technical limitations. We used combinations of transgenic reporter systems, recombinase activated viral vectors, quantitative translatome analysis, and high sensitivity in situ hybridization to identify D2 receptor expressing cells and establish a map of their respective projections. Our results identified previously uncharacterized clusters of D2 expressing neurons in limbic and sensory regions of the adult mouse brain cortex. Characterization of these clusters by translatome analysis and cell type specific labeling revealed highly heterogeneous expression of D2 receptors in principal neurons and various populations of interneurons across cortical areas. Transcript enrichment analysis also demonstrated variable levels of D2 receptor expression and several orphan G-protein-coupled receptors coexpression in different neuronal clusters, thus suggesting strategies for genetic and therapeutic targeting of D2 expressing neurons in specific cortical areas. These results pave the way for a thorough re-examination of cortical D2 receptor functions, which could provide information about neuronal circuits involved in psychotic and mood disorders.

Comprehensive Genome-Wide Approaches to Activity-Dependent Translational Control in Neurons.

Activity-dependent regulation of gene expression is critical in experience-mediated changes in the brain. Although less appreciated than transcriptional control, translational control is a crucial regulatory step of activity-mediated gene expression in physiological and pathological conditions. In the first part of this review, we overview evidence demonstrating the importance of translational controls under the context of synaptic plasticity as well as learning and memory. Then, molecular mechanisms underlying the translational control, including post-translational modifications of translation factors, mTOR signaling pathway, and local translation, are explored. We also summarize how activity-dependent translational regulation is associated with neurodevelopmental and psychiatric disorders, such as autism spectrum disorder and depression. In the second part, we highlight how recent application of high-throughput sequencing techniques has added insight into genome-wide studies on translational regulation of neuronal genes. Sequencing-based strategies to identify molecular signatures of the active neuronal population responding to a specific stimulus are discussed. Overall, this review aims to highlight the implication of translational control for neuronal gene regulation and functions of the brain and to suggest prospects provided by the leading-edge techniques to study yet-unappreciated translational regulation in the nervous system.

Fetal origin confers radioresistance on liver macrophages via p21cip1/WAF1.

BACKGROUND & AIMS: Cells of hematopoietic origin, including macrophages, are generally radiation sensitive, but a subset of Kupffer cells (KCs) is relatively radioresistant. Here, we focused on the identity of the radioresistant KCs in unmanipulated mice and the mechanism of radioresistance. METHODS: We employed Emr1- and inducible CX3Cr1-based fate-mapping strategies combined with the RiboTag reporter to identify the total KCs and the embryo-derived KCs, respectively. The KC compartment was reconstituted with adult bone-marrow-derived KCs (bm-KCs) using clodronate depletion. Mice were lethally irradiated and transplanted with donor bone marrow, and the radioresistance of bone-marrow- or embryo-derived KCs was studied. Gene expression was analyzed using in situ mRNA isolation via RiboTag reporter mice, and the translatomes were compared among subsets. RESULTS: Here, we identified the radioresistant KCs as the long-lived subset that is derived from CX3CR1-expressing progenitor cells in fetal life, while adult bm-KCs do not resist irradiation. While both subsets upregulated the Cdkn1a gene, encoding p21-cip1/WAF1 protein, radioresistant embryo-derived KCs showed a greater increase in response to irradiation. In the absence of this molecule, the radioresistance of KCs was compromised. Replacement KCs, derived from adult hematopoietic stem cells, differed from radioresistant KCs in their expression of genes related to immunity and phagocytosis. CONCLUSIONS: Here, we show that, in the murine liver, a subset of KCs of embryonic origin resists lethal irradiation through Cdkn1a upregulation and is maintained for a long period, while bm-KCs do not survive lethal irradiation. LAY SUMMARY: Kupffer cells (KCs) are the tissue-resident macrophages of the liver. KCs can be originated from fetal precursors and from monocytes during the fetal stage and post-birth, respectively. Most immune cells in mice are sensitive to lethal-irradiation-induced death, while a subset of KCs resists radiation-induced death. These radioresistant KCs continue to live in the irradiated mice. We discovered that this relatively radioresistant KC subset are the fetal-derived KCs, and they achieve this through cell-cycle arrest. Understanding the radiobiology of KCs will provide valuable insights into the mechanisms that elicit radiation-induced liver disease.

Different Responses to a High-Fat Diet in IL-6 Conditional Knockout Mice Driven by Constitutive GFAP-Cre and Synapsin 1-Cre Expression.

BACKGROUND/AIMS: Interleukin-6 (IL-6) is a major cytokine controlling body weight and metabolism, at least in part through actions in the central nervous system (CNS) from local sources. METHODS: We herewith report results obtained in conditional IL-6 KO mice for brain cells (Il6ΔGfap and Il6ΔSyn). RESULTS: The reporter RiboTag mouse line demonstrated specific astrocytic expression of GFAP-dependent Cre in the hypothalamus but not in other brain areas, whereas that of synapsin 1-dependent Cre was specific for neurons. Feeding a high-fat diet (HFD) or a control diet showed that Il6ΔGfap and Il6ΔSyn mice were more prone and resistant, respectively, to HFD-induced obesity. Energy intake was not altered in HFD experiments, but it was reduced in Il6ΔSyn male mice following a 24-h fast. HFD increased circulating insulin, leptin, and cholesterol levels, decreased triglycerides, and caused impaired responses to the insulin and glucose tolerance tests. In Il6ΔGfap mice, the only significant difference observed was an increase in insulin levels of females, whereas in Il6ΔSyn mice the effects of HFD were decreased. Hypothalamic Agrp expression was significantly decreased by HFD, further decreased in Il6ΔGfap, and increased in Il6ΔSyn female mice. Hypothalamic Il-6 mRNA levels were not decreased in Il6ΔSyn mice and even increased in Il6ΔGfapmale mice. Microarray analysis of hypothalamic RNA showed that female Il6ΔGfap mice had increased interferon-related pathways and affected processes in behavior, modulation of chemical synaptic transmission, learning, and memory. CONCLUSION: The present results demonstrate that brain production of IL-6 regulates body weight in the context of caloric excess and that the cellular source is critical.

A comparative analysis of library prep approaches for sequencing low input translatome samples.

BACKGROUND: Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. RESULTS: We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. CONCLUSION: RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.

Opposing role for Egr3 in nucleus accumbens cell subtypes in cocaine action.

An imbalance in molecular signaling cascades and transcriptional regulation in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes, those enriched in dopamine D1 versus D2 receptors, is implicated in the behavioral responses to psychostimulants. To provide further insight into the molecular mechanisms occurring in MSN subtypes by cocaine, we examined the transcription factor early growth response 3 (Egr3). We evaluated Egr3 because it is a target of critical cocaine-mediated signaling pathways and because Egr3-binding sites are found on promoters of key cocaine-associated molecules. We first used a RiboTag approach to obtain ribosome-associated transcriptomes from each MSN subtype and found that repeated cocaine administration induced Egr3 ribosome-associated mRNA in NAc D1-MSNs while reducing Egr3 in D2-MSNs. Using Cre-inducible adeno-associated viruses combined with D1-Cre and D2-Cre mouse lines, we observed that Egr3 overexpression in D1-MSNs enhances rewarding and locomotor responses to cocaine, whereas overexpression in D2-MSNs blunts these behaviors. miRNA knock-down of Egr3 in MSN subtypes produced opposite behavioral responses from those observed with overexpression. Finally, we found that repeated cocaine administration altered Egr3 binding to promoters of genes that are important for cocaine-mediated cellular and behavioral plasticity. Genes with increased Egr3 binding to promoters, Camk2α, CREB, FosB, Nr4a2, and Sirt1, displayed increased mRNA in D1-MSNs and, in some cases, a reduction in D2-MSNs. Histone and the DNA methylation enzymes G9a and Dnmt3a displayed reduced Egr3 binding to their promoters and reduced mRNA in D1-MSNs. Our study provides novel insight into an opposing role of Egr3 in select NAc MSN subtypes in cocaine action.

drd2-cre:ribotag mouse line unravels the possible diversity of dopamine d2 receptor-expressing cells of the dorsal mouse hippocampus.

Increasing evidences suggest that dopamine facilitates the encoding of novel memories by the hippocampus. However, the role of dopamine D2 receptors (D2R) in such regulations remains elusive due to the lack of the precise identification of hippocampal D2R-expressing cells. To address this issue, mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope were crossed with Drd2-Cre mice allowing the selective expression of HA in D2R-containing cells (Drd2-Cre:RiboTag mice). This new transgenic model revealed a more widespread pattern of D2R-expressing cells identified by HA immunoreactivity than the one initially reported in Drd2-EGFP mice, in which the hilar mossy cells were the main neuronal population detectable. In Drd2-Cre:RiboTag mice, scattered HA/GAD67-positive neurons were detected throughout the CA1/CA3 subfields, being preferentially localized in stratum oriens and stratum lacunosum-moleculare. At the cellular level, HA-labeled cells located in CA1/CA3 subfields co-localized with calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (neuropeptide Y, somatostatin), and other markers (neuronal nitric oxide synthase, mGluR1α, reelin, coupTFII, and potassium channel-interacting protein 1). These results suggest that in addition to the glutamatergic hilar mossy cells, D2R-expressing cells constitute a subpopulation of GABAergic hippocampal interneurons.

Investigating cocaine- and abstinence-induced effects on astrocyte gene expression in the nucleus accumbens.

In recent years, astrocytes have been increasingly implicated in cellular mechanisms of substance use disorders (SUD). Astrocytes are structurally altered following exposure to drugs of abuse; specifically, astrocytes within the nucleus accumbens (NAc) exhibit significantly decreased surface area, volume, and synaptic colocalization after operant self-administration of cocaine and extinction or protracted abstinence (45 days). However, the mechanisms that elicit these morphological modifications are unknown. The current study aims to elucidate the molecular modifications that lead to observed astrocyte structural changes in rats across cocaine abstinence using astrocyte-specific RiboTag and RNA-seq, as an unbiased, comprehensive approach to identify genes whose transcription or translation change within NAc astrocytes following cocaine self-administration and extended abstinence. Using this method, our data reveal cellular processes including cholesterol biosynthesis that are altered specifically by cocaine self-administration and abstinence, suggesting that astrocyte involvement in these processes is changed in cocaine-abstinent rats. Overall, the results of this study provide insight into astrocyte functional adaptations that occur due to cocaine exposure or during cocaine withdrawal, which may pinpoint further mechanisms that contribute to cocaine-seeking behavior.

Translatome Profiling of Tissue-Resident Macrophages Using the RiboTag Approach.

Global gene expression profiling has provided valuable insights into the specific contributions of different cell types to various physiological processes. Notably though, both bulk and single-cell transcriptomics require the prior retrieval of the cells from their tissue context to be analyzed. Isolation protocols for tissue macrophages are, however, notoriously inefficient and, moreover, prone to introduce considerable bias and artifacts. Here, we will discuss a valuable alternative, originally introduced by Amieux and colleagues. This so-called RiboTag approach allows, in combination with respective macrophage-specific Cre transgenic lines, to retrieve macrophage translatomes from crude tissue extracts. We will review our experience with this ingenious method, focusing on the study of brain macrophages, including microglia and border-associated cells. We will elaborate on the advantages of the RiboTag approach that render it a valuable complement to standard cell sorting-based profiling strategies, especially for the investigation of tissue macrophages.

Selective Vulnerability to Neurodegenerative Disease: Insights from Cell Type-Specific Translatome Studies.

Neurodegenerative diseases (NDs) manifest a wide variety of clinical symptoms depending on the affected brain regions. Gaining insights into why certain regions are resistant while others are susceptible is vital for advancing therapeutic strategies. While gene expression changes offer clues about disease responses across brain regions, the mixture of cell types therein obscures experimental results. In recent years, methods that analyze the transcriptomes of individual cells (e.g., single-cell RNA sequencing or scRNAseq) have been widely used and have provided invaluable insights into specific cell types. Concurrently, transgene-based techniques that dissect cell type-specific translatomes (CSTs) in model systems, like RiboTag and bacTRAP, offer unique advantages but have received less attention. This review juxtaposes the merits and drawbacks of both methodologies, focusing on the use of CSTs in understanding conditions like amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), and specific prion diseases like fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and acquired prion disease. We conclude by discussing the emerging trends observed across multiple diseases and emerging methods.

The molecular phenotype of kisspeptin neurons in the medial amygdala of female mice.

Reproduction is regulated through the hypothalamic-pituitary-gonadal (HPG) axis, largely via the action of kisspeptin neurons in the hypothalamus. Importantly, Kiss1 neurons have been identified in other brain regions, including the medial amygdala (MeA). Though the MeA is implicated in regulating aspects of both reproductive physiology and behavior, as well as non-reproductive processes, the functional roles of MeA Kiss1 neurons are largely unknown. Additionally, besides their stimulation by estrogen, little is known about how MeA Kiss1 neurons are regulated. Using a RiboTag mouse model in conjunction with RNA-seq, we examined the molecular profile of MeA Kiss1 neurons to identify transcripts that are co-expressed in MeA Kiss1 neurons of female mice and whether these transcripts are modulated by estradiol (E2) treatment. RNA-seq identified >13,800 gene transcripts co-expressed in female MeA Kiss1 neurons, including genes for neuropeptides and receptors implicated in reproduction, metabolism, and other neuroendocrine functions. Of the >13,800 genes co-expressed in MeA Kiss1 neurons, only 45 genes demonstrated significantly different expression levels due to E2 treatment. Gene transcripts such as Kiss1, Gal, and Oxtr increased in response to E2 treatment, while fewer transcripts, such as Esr1 and Cyp26b1, were downregulated by E2. Dual RNAscope and immunohistochemistry was performed to validate co-expression of MeA Kiss1 with Cck and Cartpt. These results are the first to establish a profile of genes actively expressed by MeA Kiss1 neurons, including a subset of genes regulated by E2, which provides a useful foundation for future investigations into the regulation and function of MeA Kiss1 neurons.

Rapid isolation of intact retinal astrocytes: a novel approach.

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.

Spatiomolecular Characterization of Dopamine D2 Receptors Cells in the Mouse External Globus Pallidus.

The external globus pallidus (GPe) is part of the basal ganglia circuit and plays a key role in controlling the actions. Although, many evidence indicate that dopamine through its activation of D2 receptors (D2Rs) modulates the GPe neuronal activity, the precise spatiomolecular characterization of cell populations expressing D2Rs in the mouse GPe is still lacking. By combining single molecule in situ hybridization, cell type-specific imaging analyses, and electrophysiology slice recordings, we found that GPe D2R cells are neurons preferentially localized in the caudal portion of GPe. These neu- rons comprising pallido-striatal, pallido-nigral, and pallido-cortical neurons segregate into two distinct populations displaying molecular and electrophysiological features of GPe GABAergic PV/NKX2.1 and cholinergic neurons respectively. By clarifying the spatial molecular identity of GPe D2R neurons in the mouse, this work provides the basis for future studies aiming at disentangling the action of do- pamine within the GPe.