Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium.

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Particulate levodopa nose-to-brain delivery targets dopamine to the brain with no plasma exposure.

Levodopa (L-DOPA) is an oral Parkinson's Disease drug that generates the active metabolite - dopamine (DA) in vivo. However, oral L-DOPA exhibits low oral bioavailability, limited brain uptake, peripheral DA-mediated side effects and its poor brain bioavailability can lead to long-term complications. Here we show that L-DOPA forms stable (for at least 5 months) 300 nm nanoparticles when encapsulated within N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ). A nano-in-microparticle GCPQ-L-DOPA formulation (D50 = 7.2 µm), prepared by spray-drying, was stable for one month when stored at room and refrigeration temperatures and was capable of producing the original GCPQ-L-DOPA nanoparticles upon aqueous reconstitution. Nasal administration of reconstituted GCPQ-L-DOPA nanoparticles to rats resulted in significantly higher DA levels in the brain (Cmax of 94 ng g-1 above baseline levels 2 h post-dosing) when compared to nasal administration of L-DOPA alone, with DA being undetectable in the brain with the latter. Furthermore, nasal GCPQ-L-DOPA resulted in higher levels of L-DOPA in the plasma (a 17-fold increase in the Cmax, when compared to L-DOPA alone) with DA undetectable in the plasma from both formulations. These data provide evidence of effective delivery of DA to the brain with the GCPQ-L-DOPA formulation.

Astrocytes in cocaine addiction and beyond.

Drug addiction remains a key biomedical challenge facing current neuroscience research. In addition to neural mechanisms, the focus of the vast majority of studies to date, astrocytes have been increasingly recognized as an "accomplice." According to the tripartite synapse model, astrocytes critically regulate nearby pre- and postsynaptic neuronal substrates to craft experience-dependent synaptic plasticity, including synapse formation and elimination. Astrocytes within brain regions that are implicated in drug addiction exhibit dynamic changes in activity upon exposure to cocaine and subsequently undergo adaptive changes themselves during chronic drug exposure. Recent results have identified several key astrocytic signaling pathways that are involved in cocaine-induced synaptic and circuit adaptations. In this review, we provide a brief overview of the role of astrocytes in regulating synaptic transmission and neuronal function, and discuss how cocaine influences these astrocyte-mediated mechanisms to induce persistent synaptic and circuit alterations that promote cocaine seeking and relapse. We also consider the therapeutic potential of targeting astrocytic substrates to ameliorate drug-induced neuroplasticity for behavioral benefits. While primarily focusing on cocaine-induced astrocytic responses, we also include brief discussion of other drugs of abuse where data are available.

Cocaine Triggers Astrocyte-Mediated Synaptogenesis.

BACKGROUND: Synaptogenesis is essential in forming new neurocircuits during development, and this is mediated in part by astrocyte-released thrombospondins (TSPs) and activation of their neuronal receptor, α2δ-1. Here, we show that this developmental synaptogenic mechanism is utilized during cocaine experience to induce spinogenesis and the generation of AMPA receptor-silent glutamatergic synapses in the adult nucleus accumbens shell (NAcSh). METHODS: Using multidisciplinary approaches including astrocyte Ca2+ imaging, genetic mouse lines, viral-mediated gene transfer, and operant behavioral procedures, we monitor the response of NAcSh astrocytes to cocaine administration and examine the role of astrocytic TSP-α2δ-1 signaling in cocaine-induced silent synapse generation as well as the behavioral impact of astrocyte-mediated synaptogenesis and silent synapse generation. RESULTS: Cocaine administration acutely increases Ca2+ events in NAcSh astrocytes, while decreasing astrocytic Ca2+ blocks cocaine-induced generation of silent synapses. Furthermore, knockout of TSP2, or pharmacological inhibition or viral-mediated knockdown of α2δ-1, prevents cocaine-induced generation of silent synapses. Moreover, disrupting TSP2-α2δ-1-mediated spinogenesis and synapse generation in NAcSh decreases cue-induced cocaine seeking after withdrawal from cocaine self-administration and cue-induced reinstatement of cocaine seeking after drug extinction. CONCLUSIONS: These results establish that silent synapses are generated by an astrocyte-mediated synaptogenic mechanism in response to cocaine experience and embed critical cue-associated memory traces that promote cocaine relapse.

Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in HLA-A*33:03, HLA-A*68:01, HLA-B*14:02 and HLA-B*40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.

The Stem Cell Marker Lgr5 Defines a Subset of Postmitotic Neurons in the Olfactory Bulb.

Lgr5, leucine-rich repeat-containing G-protein coupled receptor 5, is a bona fide biomarker for stem cells in multiple tissues. Lgr5 is also expressed in the brain, but the identities and properties of these Lgr5+ cells are still elusive. Using an Lgr5-EGFP reporter mouse line, we found that, from early development to adulthood, Lgr5 is highly expressed in the olfactory bulb (OB), an area with ongoing neurogenesis. Immunostaining with stem cell, glial, and neuronal markers reveals that Lgr5 does not label stem cells in the OB but instead labels a heterogeneous population of neurons with preference in certain subtypes. Patch-clamp recordings in OB slices reveal that Lgr5-EGFP+ cells fire action potentials and display spontaneous excitatory postsynaptic events, indicating that these neurons are integrated into OB circuits. Interestingly, R-spondin 3, a potential ligand of Lgr5, is also expressed in the adult OB. Collectively, our data indicate that Lgr5-expressing cells in the OB are fully differentiated neurons and imply distinct roles of Lgr5 and its ligand in postmitotic cells.SIGNIFICANCE STATEMENT Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) is a bona fide stem cell marker in many body organs. Here we report that Lgr5 is also highly expressed in the olfactory bulb (OB), the first relay station in the brain for processing odor information and one of the few neural structures that undergo continuous neurogenesis. Surprisingly, Lgr5 is not expressed in the OB stem cells, but instead in a few subtypes of terminally differentiated neurons, which are incorporated into the OB circuit. This study reveals that Lgr5+ cells in the brain represent a nonstem cell lineage, implying distinct roles of Lgr5 in postmitotic neurons.