Roman A. Romanov

Afferent neurotransmission mediated by hemichannels in mammalian taste cells

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor. Among electrophysiologically distinguishable taste cells, a population was found that released ATP in a manner that was Ca2+ independent but voltage‐dependent. Data from physiological and pharmacological experiments suggested that ATP was released from taste cells via specific channels, likely to be connexin or pannexin hemichannels. A small fraction of ATP‐secreting taste cells responded to bitter compounds, indicating that they express taste receptors, their G‐protein‐coupled and downstream transduction elements. Single cell RT–PCR revealed that ATP‐secreting taste cells expressed gustducin, TRPM5, PLCβ2, multiple connexins and pannexin 1. Altogether, our data indicate that tastant‐responsive taste cells release the neurotransmitter ATP via a non‐exocytotic mechanism dependent upon the generation of an action potential.

Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system

One size does not fit all Oligodendrocytes are best known for their ability to myelinate brain neurons, thus increasing the speed of signal transmission. Marques et al. surveyed oligodendrocytes of developing mice and found unexpected heterogeneity. Transcriptional analysis identified 12 populations, ranging from precursors to mature oligodendrocytes. Transcriptional profiles diverged as the oligodendrocytes matured, building distinct populations. One population was responsive to motor learning, and another, with a different transcriptome, traveled along blood vessels. Science, this issue p. 1326 Brain oligodendrocytes express transcriptional heterogeneity between brain regions and age of development. Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra+ oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra+ population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Summary Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Voltage dependence of ATP secretion in mammalian taste cells.

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable.

Summary Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.

Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells

Three types of morphologically and functionally distinct taste cells operate in the mammalian taste bud. We demonstrate here the expression of two G-protein-coupled receptors from the family C, CASR and GPRC6A, in the taste tissue and identify transcripts for both receptors in type I cells, no transcripts in type II cells and only CASR transcripts in type III cells, by using the SMART-PCR RNA amplification method at the level of individual taste cells. Type I taste cells responded to calcimimetic NPS R-568, a stereoselective CASR probe, with Ca2+ transients, whereas type I and type II cells were not specifically responsive. Consistent with these findings, certain amino acids stimulated PLC-dependent Ca2+ signaling in type III cells, but not in type I and type II cells, showing the following order of efficacies: Phe~Glu>Arg. Thus, CASR is coupled to Ca2+ mobilization solely in type III cells. CASR was cloned from the circumvallate papilla into a pIRES2-EGFP plasmid and heterologously expressed in HEK-293 cells. The transfection with CASR enabled HEK-293 cells to generate Ca2+ transients in response to the amino acids, of which, Phe was most potent. This observation and some other facts favor CASR as the predominant receptor subtype endowing type III cells with the ability to detect amino acids. Altogether, our results indicate that type III cells can serve a novel chemosensory function by expressing the polymodal receptor CASR. A role for CASR and GPRC6A in physiology of taste cells of the type I remains to be unveiled.

Electrophysiologically identified subpopulations of taste bud cells

The heterogeneous population of mammalian taste cells includes several cellular subtypes specializing in distinct physiological functions. They are poorly understood at the single cell level because the available physiological data have generally been obtained from unidentified taste cells. We recorded them from individual taste cells isolated from circumvallate, foliate, and fungiform papilla of the mouse, employing the patch clamp technique, and tried to elucidate whether universal electrophysiological criteria may be established for the identification of functionally different cellular subpopulations. It was found that irrespective of the papillae type, most ( approximately 96%) of robust taste cells could be categorized into three distinct subgroups on the basis of families of whole-cell (WC) currents exhibited in response to membrane polarization. The validity of this quite simple criterion was further confirmed by using different voltage clamp protocols, ion substitutions, and channel blockers to record different ionic currents, including voltage-gated (VG) Ca(2+), inward-rectifying K(+), and hyperpolarization-activated currents. Given that our findings are based on the statistically significant number of recordings, we believe that the electrophysiological identification of taste cells presented here may be effective for further studies on single taste cell physiology, including taste transduction.

Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model

Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for Parkinsons disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinsons disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinsons disease by delivery of genes rather than cells.

A secretagogin locus of the mammalian hypothalamus controls stress hormone release

A hierarchical hormonal cascade along the hypothalamic‐pituitary‐adrenal axis orchestrates bodily responses to stress. Although corticotropin‐releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single‐cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH‐releasing neuron population reliant on secretagogins Ca2+ sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogins loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.