Magnetogenetic cell activation using endogenous ferritin.

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro . In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo . Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo . Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells.

The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that ß-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration.

Author Correction: Microbiota modulate sympathetic neurons via a gut-brain circuit.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microbiota modulate sympathetic neurons via a gut-brain circuit.

Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content1, regulating both physiological intestinal functions such as nutrient absorption and motility2,3, and brain-wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology4. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.

A leptin-BDNF pathway regulating sympathetic innervation of adipose tissue.

Mutations in the leptin gene (ob) result in a metabolic disorder that includes severe obesity1, and defects in thermogenesis2 and lipolysis3, both of which are adipose tissue functions regulated by the sympathetic nervous system. However, the basis of these sympathetic-associated abnormalities remains unclear. Furthermore, chronic leptin administration reverses these abnormalities in adipose tissue, but the underlying mechanism remains to be discovered. Here we report that ob/ob mice, as well as leptin-resistant diet-induced obese mice, show significant reductions of sympathetic innervation of subcutaneous white and brown adipose tissue. Chronic leptin treatment of ob/ob mice restores adipose tissue sympathetic innervation, which in turn is necessary to correct the associated functional defects. The effects of leptin on innervation are mediated via agouti-related peptide and pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus. Deletion of the gene encoding the leptin receptor in either population leads to reduced innervation in fat. These agouti-related peptide and pro-opiomelanocortin neurons act via brain-derived neurotropic factor-expressing neurons in the paraventricular nucleus of the hypothalamus (BDNFPVH). Deletion of BDNFPVH blunts the effects of leptin on innervation. These data show that leptin signalling regulates the plasticity of sympathetic architecture of adipose tissue via a top-down neural pathway that is crucial for energy homeostasis.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.

We previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5'-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12-/- and CalDAG GEFI-/- mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.