Cell-type specialization is encoded by specific chromatin topologies.

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Reproducing the dopamine pathophysiology of schizophrenia and approaches to ameliorate it: a translational imaging study with ketamine.

Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. The mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we develop a translational mouse model of the dopamine pathophysiology seen in schizophrenia and test approaches to reverse the dopamine changes. Mice were treated with sub-chronic ketamine (30 mg/kg) or saline and then received in vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open-field test. In vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen's d = 2.5) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of PV interneurons in pre-limbic cortex and ventral subiculum of the hippocampus. Sub-chronic ketamine reduced PV expression in these cortical and hippocampal regions. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1) and 5-HT1A receptors but no appreciable action at dopamine D2 receptors, significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, that this requires activation of midbrain dopamine neurons, and can be ameliorated by activating PV interneurons and by a TAAR1/5-HT1A agonist. This identifies novel therapeutic approaches for targeting presynaptic dopamine dysfunction in patients with schizophrenia and effects of ketamine relevant to its therapeutic use for  treating major depression.

Assessing the Value of Cemiplimab for Adults With Advanced Cutaneous Squamous Cell Carcinoma: A Cost-Effectiveness Analysis.

OBJECTIVES: To evaluate the cost-effectiveness of cemiplimab in patients with advanced cutaneous squamous cell carcinoma (CSCC) from a payer perspective in the United States. METHODS: A partitioned survival model was developed to assess the cost-effectiveness of cemiplimab versus historical standard of care (SOC). All inputs were identified based on a systematic literature review, supplemented by expert opinion where necessary. Clinical inputs for cemiplimab were based on individual patient data from a cemiplimab phase 2 single-arm trial (NCT27060498). For SOC, analysis was based on a pooled analysis of single-arm clinical trials and retrospective studies evaluating chemotherapy and epidermal growth factor receptor inhibitors (cetuximab, erlotinib, and gefitinib) identified via a systematic literature review (6 of the 27 included studies). Overall survival and progression-free survival were extrapolated over a lifetime horizon. Costs were included for drug acquisition, drug administration, management of adverse events, subsequent therapy, disease management, and terminal care. Unit costs were based on published 2019 US list prices. RESULTS: In the base case, cemiplimab versus SOC resulted in an incremental cost-effectiveness ratio of $99 447 per quality adjusted-life year (QALY), where incremental costs and QALYs were $372 108 and 3.74, respectively. At a willingness-to-pay threshold of $150 000/QALY, the probabilistic sensitivity analysis suggests a 90% probability that cemiplimab is cost-effective compared to SOC. Scenario analyses resulted in incremental cost-effectiveness ratios ranging from $90 590 to $148 738. CONCLUSIONS: Compared with historical SOC, cemiplimab is a cost-effective use of US payer resources for the treatment of advanced CSCC and is expected to provide value for money.

Plasma and cerebrospinal fluid ABeta42 for the differential diagnosis of Alzheimer's disease dementia in participants diagnosed with any dementia subtype in a specialist care setting.

BACKGROUND: Dementia is a syndrome that comprises many differing pathologies, including Alzheimer's disease dementia (ADD), vascular dementia (VaD) and frontotemporal dementia (FTD). People may benefit from knowing the type of dementia they live with, as this could inform prognosis and may allow for tailored treatment. Beta-amyloid (1-42) (ABeta42) is a protein which decreases in both the plasma and cerebrospinal fluid (CSF) of people living with ADD, when compared to people with no dementia. However, it is not clear if changes in ABeta42 are specific to ADD or if they are also seen in other types of dementia. It is possible that ABeta42 could help differentiate ADD from other dementia subtypes. OBJECTIVES: To determine the accuracy of plasma and CSF ABeta42 for distinguishing ADD from other dementia subtypes in people who meet the criteria for a dementia syndrome. SEARCH METHODS: We searched MEDLINE, and nine other databases up to 18 February 2020. We checked reference lists of any relevant systematic reviews to identify additional studies. SELECTION CRITERIA: We considered cross-sectional studies that differentiated people with ADD from other dementia subtypes. Eligible studies required measurement of participant plasma or CSF ABeta42 levels and clinical assessment for dementia subtype. DATA COLLECTION AND ANALYSIS: Seven review authors working independently screened the titles and abstracts generated by the searches. We collected data on study characteristics and test accuracy. We used the second version of the 'Quality Assessment of Diagnostic Accuracy Studies' (QUADAS-2) tool to assess internal and external validity of results. We extracted data into 2 x 2 tables, cross-tabulating index test results (ABeta42) with the reference standard (diagnostic criteria for each dementia subtype). We performed meta-analyses using bivariate, random-effects models. We calculated pooled estimates of sensitivity, specificity, positive predictive values, positive and negative likelihood ratios, and corresponding 95% confidence intervals (CIs). In the primary analysis, we assessed accuracy of plasma or CSF ABeta42 for distinguishing ADD from other mixed dementia types (non-ADD). We then assessed accuracy of ABeta42 for differentiating ADD from specific dementia types: VaD, FTD, dementia with Lewy bodies (DLB), alcohol-related cognitive disorder (ARCD), Creutzfeldt-Jakob disease (CJD) and normal pressure hydrocephalus (NPH). To determine test-positive cases, we used the ABeta42 thresholds employed in the respective primary studies. We then performed sensitivity analyses restricted to those studies that used common thresholds for ABeta42. MAIN RESULTS: We identified 39 studies (5000 participants) that used CSF ABeta42 levels to differentiate ADD from other subtypes of dementia. No studies of plasma ABeta42 met the inclusion criteria. No studies were rated as low risk of bias across all QUADAS-2 domains. High risk of bias was found predominantly in the domains of patient selection (28 studies) and index test (25 studies). The pooled estimates for differentiating ADD from other dementia subtypes were as follows: ADD from non-ADD: sensitivity 79% (95% CI 0.73 to 0.85), specificity 60% (95% CI 0.52 to 0.67), 13 studies, 1704 participants, 880 participants with ADD; ADD from VaD: sensitivity 79% (95% CI 0.75 to 0.83), specificity 69% (95% CI 0.55 to 0.81), 11 studies, 1151 participants, 941 participants with ADD; ADD from FTD: sensitivity 85% (95% CI 0.79 to 0.89), specificity 72% (95% CI 0.55 to 0.84), 17 studies, 1948 participants, 1371 participants with ADD; ADD from DLB: sensitivity 76% (95% CI 0.69 to 0.82), specificity 67% (95% CI 0.52 to 0.79), nine studies, 1929 participants, 1521 participants with ADD. Across all dementia subtypes, sensitivity was greater than specificity, and the balance of sensitivity and specificity was dependent on the threshold used to define test positivity. AUTHORS' CONCLUSIONS: Our review indicates that measuring ABeta42 levels in CSF may help differentiate ADD from other dementia subtypes, but the test is imperfect and tends to misdiagnose those with non-ADD as having ADD. We would caution against the use of CSF ABeta42 alone for dementia classification. However, ABeta42 may have value as an adjunct to a full clinical assessment, to aid dementia diagnosis.

Transcriptional profiling aligned with in situ expression image analysis reveals mosaically expressed molecular markers for GABA neuron sub-groups in the ventral tegmental area.

γ-Aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide local inhibitory control of dopamine neuron activity and send long-range projections to several target regions including the nucleus accumbens. They play diverse roles in reward and aversion, suggesting that they be comprised of several functionally distinct sub-groups, but our understanding of this diversity has been limited by a lack of molecular markers that might provide genetic entry points for cell type-specific investigations. To address this, we conducted transcriptional profiling of GABA neurons and dopamine neurons using immunoprecipitation of tagged polyribosomes (RiboTag) and RNAseq. First, we directly compared these two transcriptomes in order to obtain a list of genes enriched in GABA neurons compared with dopamine neurons. Next, we created a novel bioinformatic approach, that used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) gene ontology database and VTA gene expression data from the Allen Mouse Brain Atlas, from which we obtained 6 candidate genes: Cbln4, Rxfp3, Rora, Gpr101, Trh and Nrp2. As a final step, we verified the selective expression of these candidate genes in sub-groups of GABA neurons in the VTA (and neighbouring substantia nigra pars compacta) using immunolabelling. Taken together, our study provides a valuable toolbox for the future investigation of GABA neuron sub-groups in the VTA.

A single dose of cocaine rewires the 3D genome structure of midbrain dopamine neurons.

Midbrain dopamine neurons (DNs) respond to a first exposure to addictive drugs and play key roles in chronic drug usage1-3. As the synaptic and transcriptional changes that follow an acute cocaine exposure are mostly resolved within a few days4,5, the molecular changes that encode the long-term cellular memory of the exposure within DNs remain unknown. To investigate whether a single cocaine exposure induces long-term changes in the 3D genome structure of DNs, we applied Genome Architecture Mapping and single nucleus transcriptomic analyses in the mouse midbrain. We found extensive rewiring of 3D genome architecture at 24 hours past exposure which remains or worsens by 14 days, outlasting transcriptional responses. The cocaine-induced chromatin rewiring occurs at all genomic scales and affects genes with major roles in cocaine-induced synaptic changes. A single cocaine exposure triggers extensive long-lasting changes in chromatin condensation in post-synaptic and post-transcriptional regulatory genes, for example the unfolding of Rbfox1 which becomes most prominent 14 days post exposure. Finally, structurally remodeled genes are most expressed in a specific DN sub-type characterized by low expression of the dopamine auto-receptor Drd2, a key feature of highly cocaine-sensitive cells. These results reveal an important role for long-lasting 3D genome remodelling in the cellular memory of a single cocaine exposure, providing new hypotheses for understanding the inception of drug addiction and 3D genome plasticity.

The PCI domains are "winged" HEAT domains.

The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members: huntingtin, eukaryotic translation elongation factor 3 (eEF3), protein phosphatase 2 regulatory A subunit (PP2A), and mechanistic target of rapamycin (mTOR). HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a "winged-helix" (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the 26S Proteasome "lid" regulatory subcomplex, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). We noted that in structure similarity searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group, as currently thought. Here, we report extensive structure similarity analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural similarity with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated self-association into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3.

Regulation of the interactions between human eIF5 and eIF1A by the CK2 kinase.

Translation initiation in eukaryotes relies on a complex network of interactions that are continuously reorganized throughout the process. As more information becomes available about the structure of the ribosomal preinitiation complex (PIC) at various points in translation initiation, new questions arise about which interactions occur when, their roles, and regulation. The eukaryotic translation factor (eIF) 5 is the GTPase-activating protein (GAP) for the GTPase eIF2, which brings the initiator Met-tRNAi to the PIC. eIF5 also plays a central role in PIC assembly and remodeling through interactions with other proteins, including eIFs 1, 1A, and 3c. Phosphorylation by casein kinase 2 (CK2) significantly increases the eIF5 affinity for eIF2. The interaction between eIF5 and eIF1A was reported to be mediated by the eIF5 C-terminal domain (CTD) and the eIF1A N-terminal tail. Here, we report a new contact interface, between eIF5-CTD and the oligonucleotide/oligosaccharide-binding fold (OB) domain of eIF1A, which contributes to the overall affinity between the two proteins. We also show that the interaction is modulated by dynamic intramolecular interactions within both eIF5 and eIF1A. CK2 phosphorylation of eIF5 increases its affinity for eIF1A, offering new insights into the mechanisms by which CK2 stimulates protein synthesis and cell proliferation.

Dynamic interaction network involving the conserved intrinsically disordered regions in human eIF5.

Translation initiation in eukaryotes requires multiple eukaryotic translation initiation factors (eIFs) and involves continuous remodeling of the ribosomal preinitiation complex (PIC). The GTPase eIF2 brings the initiator Met-tRNAi to the PIC. Upon start codon selection and GTP hydrolysis, promoted by eIF5, eIF2-GDP is released in complex with eIF5. Here, we report that two intrinsically disordered regions (IDRs) in eIF5, the DWEAR motif and the C-terminal tail (CTT) dynamically contact the folded C-terminal domain (CTD) and compete with each other. The eIF5-CTD•CTT interaction favors eIF2ß binding to eIF5-CTD, whereas the eIF5-CTD•DWEAR interaction favors eIF1A binding, which suggests how intramolecular contact rearrangement could play a role in PIC remodeling. We show that eIF5 phosphorylation by CK2, which is known to stimulate translation and cell proliferation, significantly increases the eIF5 affinity for eIF2. Our results also indicate that the eIF2ß subunit has at least two, and likely three eIF5-binding sites.

Cost-effectiveness analysis of cemiplimab vs pembrolizumab for treatment of advanced cutaneous squamous cell carcinoma.

BACKGROUND: Most cutaneous squamous cell carcinomas (CSCCs) can be treated with surgical excision or radiation; however, approximately 1% of patients develop advanced disease. In 2018, the FDA approved cemiplimab-rwlc as the first programmed cell death-1 (PD-1) monoclonal antibody for the treatment of patients with metastatic CSCC or locally advanced CSCC who are not candidates for curative surgery or curative radiation. In June 2020, pembrolizumab, another PD-1 monoclonal antibody, was approved for the treatment of patients with recurrent or metastatic CSCC who are not candidates for curative surgery or radiation. We previously reported on the cost-effectiveness of cemiplimab vs historical standard of care for the treatment of advanced CSCC from a US perspective. OBJECTIVE: To estimate the cost-effectiveness of cemiplimab vs pembrolizumab for patients with advanced CSCC in the United States. METHODS: A "partitioned survival" framework was used to assess the cost-effectiveness of cemiplimab vs pembrolizumab. Clinical inputs were based on the most recent data cut of the phase 2 trials for cemiplimab (EMPOWER-CSCC-1; NCT02760498) and pembrolizumab (KEYNOTE-629). Progression-free survival and overall survival were extrapolated using parametric models until all patients had progressed or died. Health state utilities were derived from data collected in the EMPOWER-CSCC-1 trial. Costs included drug acquisition, drug administration, disease management, terminal care, and adverse events and were based on published 2020 US list prices. To assess model uncertainty, 1-way sensitivity and probabilistic sensitivity analyses (PSA) were conducted, alongside scenario analyses evaluating key modeling assumptions. RESULTS: In the base case, cemiplimab resulted in an incremental gain of 3.44 life-years (discounted) and incremental cost-effectiveness ratio (ICER) of $130,329 per quality-adjusted life-year (QALY) vs pembrolizumab. At a willingness-to-pay threshold of $150,000/QALY, PSA indicated a 71% probability that cemiplimab is cost-effective when compared with pembrolizumab. Scenario analysis resulted in ICERs ranging from $115,909 to $187,374. CONCLUSIONS: Findings suggest that cemiplimab is a cost-effective treatment for patients with advanced CSCC, compared with pembrolizumab. These results should be interpreted cautiously in the absence of head-to-head trials; however, in the absence of such data, these results can be used to inform health care decisions over resource allocation. DISCLOSURES: This study was supported by Regeneron Pharmaceuticals, Inc., and Sanofi. Paul, Cope, Keeping, Mojebi, and Ayers are employees of PRECISIONheor, which received funding to produce this work. Chen, Kuznik, and Xu are employees and stockholders of Regeneron Pharmaceuticals, Inc. Sasane is an employee and stockholder of Sanofi, Inc. Konidaris, Atsou, and Guyot are employees of Sanofi, Inc. The authors were responsible for all content and editorial decisions and received no honoraria related to the development of this publication.

nNOS-Expressing Neurons in the Ventral Tegmental Area and Substantia Nigra Pars Compacta.

GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.

Biological interaction of living cells with COSAN-based synthetic vesicles.

Cobaltabisdicarbollide (COSAN) [3,3'-Co(1,2-C2B9H11)2](-), is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes.