Dissociable Structural and Functional Hippocampal Outputs via Distinct Subiculum Cell Classes.

The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.

Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells.

Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.

Fasting-Mimicking Diet Promotes Ngn3-Driven ß-Cell Regeneration to Reverse Diabetes.

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing ß cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.

Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression.

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Compositional Control of Phase-Separated Cellular Bodies.

Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. The data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.

Dorsal Raphe Dopamine Neurons Represent the Experience of Social Isolation.

The motivation to seek social contact may arise from either positive or negative emotional states, as social interaction can be rewarding and social isolation can be aversive. While ventral tegmental area (VTA) dopamine (DA) neurons may mediate social reward, a cellular substrate for the negative affective state of loneliness has remained elusive. Here, we identify a functional role for DA neurons in the dorsal raphe nucleus (DRN), in which we observe synaptic changes following acute social isolation. DRN DA neurons show increased activity upon social contact following isolation, revealed by in vivo calcium imaging. Optogenetic activation of DRN DA neurons increases social preference but causes place avoidance. Furthermore, these neurons are necessary for promoting rebound sociability following an acute period of isolation. Finally, the degree to which these neurons modulate behavior is predicted by social rank, together supporting a role for DRN dopamine neurons in mediating a loneliness-like state. PAPERCLIP.

Primacy of Flexor Locomotor Pattern Revealed by Ancestral Reversion of Motor Neuron Identity.

Spinal circuits can generate locomotor output in the absence of sensory or descending input, but the principles of locomotor circuit organization remain unclear. We sought insight into these principles by considering the elaboration of locomotor circuits across evolution. The identity of limb-innervating motor neurons was reverted to a state resembling that of motor neurons that direct undulatory swimming in primitive aquatic vertebrates, permitting assessment of the role of motor neuron identity in determining locomotor pattern. Two-photon imaging was coupled with spike inference to measure locomotor firing in hundreds of motor neurons in isolated mouse spinal cords. In wild-type preparations, we observed sequential recruitment of motor neurons innervating flexor muscles controlling progressively more distal joints. Strikingly, after reversion of motor neuron identity, virtually all firing patterns became distinctly flexor like. Our findings show that motor neuron identity directs locomotor circuit wiring and indicate the evolutionary primacy of flexor pattern generation.

Hypothalamic UDP Increases in Obesity and Promotes Feeding via P2Y6-Dependent Activation of AgRP Neurons.

Activation of orexigenic AgRP-expressing neurons in the arcuate nucleus of the hypothalamus potently promotes feeding, thus defining new regulators of AgRP neuron activity could uncover potential novel targets for obesity treatment. Here, we demonstrate that AgRP neurons express the purinergic receptor 6 (P2Y6), which is activated by uridine-diphosphate (UDP). In vivo, UDP induces ERK phosphorylation and cFos expression in AgRP neurons and promotes action potential firing of these neurons in brain slice recordings. Consequently, central application of UDP promotes feeding, and this response is abrogated upon pharmacologic or genetic inhibition of P2Y6 as well as upon pharmacogenetic inhibition of AgRP neuron activity. In obese animals, hypothalamic UDP content is elevated as a consequence of increased circulating uridine concentrations. Collectively, these experiments reveal a potential regulatory pathway in obesity, where peripheral uridine increases hypothalamic UDP concentrations, which in turn can promote feeding via PY6-dependent activation of AgRP neurons.

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.

The cellular mechanisms of neuronal swelling underlying cytotoxic edema.

Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

MLL4 prepares the enhancer landscape for Foxp3 induction via chromatin looping.

MLL4 is an essential subunit of the histone H3 Lys4 (H3K4)-methylation complexes. We found that MLL4 deficiency compromised the development of regulatory T cells (Treg cells) and resulted in a substantial decrease in monomethylated H3K4 (H3K4me1) and chromatin interaction at putative gene enhancers, a considerable portion of which were not direct targets of MLL4 but were enhancers that interacted with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the enhancers not bound by MLL4 correlated with MLL4 binding at distant interacting regions. Deletion of an upstream MLL4-binding site diminished the abundance of H3K4me1 at the regulatory elements of the gene encoding the transcription factor Foxp3 that were looped to the MLL4-binding site and compromised both the thymic differentiation and the inducible differentiation of Treg cells. We found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping, which identifies a previously unknown mechanism for regulating the T cell enhancer landscape and affecting Treg cell differentiation.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.