Adrenal chromaffin cells comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the appropriate stress response. Deletion of the serotonin (5-HT) transporter (SERT) gene in mice (SERT-/- mice) or pharmacological block of SERT function in rodents and humans augments this sympathoadrenal stress response (epinephrine secretion). The prevailing assumption is that loss of CNS SERT alters central drive to the peripheral sympathetic nervous system. Adrenal chromaffin cells also prominently express SERT where it might coordinate accumulation of 5-HT for reuse in the autocrine control of stress-evoked catecholamine secretion. To help test this hypothesis, we have generated a novel mouse model with selective excision of SERT in the peripheral sympathetic nervous system (SERTΔTH), generated by crossing floxed SERT mice with tyrosine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was abolished in the adrenal gland but not perturbed in the CNS of SERTΔTH mice. SERT-mediated [3H] 5-HT uptake was unaltered in midbrain, hindbrain, and spinal cord synaptosomes, confirming transporter function was intact in the CNS. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in SERTΔTH mice, contrasting with the depleted 5-HT content in SERT-/- mice. Selective SERT excision reduced adrenal gland 5-HT content by ≈ 50% in SERTΔTH mice but had no effect on adrenal catecholamine content. This novel model confirms that SERT expressed in adrenal chromaffin cells is essential for maintaining wild-type levels of 5-HT and provides a powerful tool to help dissect the role of SERT in the sympathetic stress response.

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) disrupts extracellular matrix (ECM) homeostasis, leading to cartilage degradation. Upregulated a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 leads to cleavage of its substrate aggrecan (Acan) and is considered a hallmark of TMJOA. However, most research on ADAMTS5-Acan turnover has focused on hyaline cartilage, not fibrocartilage, which comprises the TMJ. The mandibular condylar cartilage (MCC) of the TMJ is organized in zones, and chondrocytes are arranged in axial rows, yet the molecular mechanisms required to generate the MCC zonal architecture have not been elucidated. Here, we test the hypothesis that ADAMTS5 is required for development of the TMJ MCC. Adamts5+/+ and Adamts5-/- murine TMJs were harvested at postnatal day 7 (P7), P21, 2 mo, and 6 mo of age; histomorphometrics indicated increased ECM. Immunohistochemistry and Western blots demonstrated the expanded ECM correlated with increased Acan localization in Adamts5-/- compared to Adamts5+/+. Cell volume was also decreased in the MCC of Adamts5-/- due to both a reduction in cell size and less mature hypertrophic chondrocytes. Analysis of chondrogenic maturation markers by quantitative real-time polymerase chain reaction indicated Col2a1, Col10a1, and Sox9 were significantly reduced in Adamts5-/- MCC compared to that of Adamts5+/+. The older (6 mo) Adamts5-/- MCC exhibited changes in chondrogenic cell arrangements, including clustering and chondrogenic atrophy, that correlated with early stages of TMJOA using modified Mankin scoring. These data indicate a potentially novel and critical role of ADAMTS5 for maturation of hypertrophic chondrocytes and establishment of the zonal architecture that, when disrupted, may lead to early onset of TMJOA.

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves -/- mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves -/- mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves-/- mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.

Reduced expression of GM1 and other major brain gangliosides GD1a, GD1b and GT1b have been reported in Parkinson's disease (PD) brain. Mechanisms underlying these changes are unclear but may be due to a deficit in the ganglioside biosynthetic process. The present study examined the extent to which deficits in gene expression of key biosynthetic enzymes involved in synthesis of GM1 and GD1b (B3galt4) and GD1a and GT1b (St3gal2) exist in neuromelanin-containing neurons in the PD substantia nigra (SN). In situ hybridization histochemistry was used to examine gene expression of B3GALT4 and ST3GAL2 in neuromelanin-containing neurons in the SN in 8 normal controls (61-92 yrs.) and 7 PD subjects (77-95 yrs). There was a significant decrease in both B3GALT4 and ST3GAL2 gene expression in residual neuromelanin-containing cells in the SN of PD patients compared to age-matched neurologically normal controls. These changes appeared to be cell-type specific as abundant B3GALT4 and ST3GAL2 gene expression was observed in non-neuromelanin containing neurons located outside of the SN in the PD brain. These data show that residual neuromelanin-containing neurons in the PD SN have decreased expression of the ganglioside biosynthetic genes B3GALT4 and ST3GAL2, consistent with previous reports of decreased levels of gangliosides GM1, GD1a, GD1b and GT1b in the PD SN. These changes may increase the vulnerability of these neurons to degeneration in response to a variety of potential stressors.

Primary familial brain calcification (PFBC) is a genetically heterogeneous disorder characterized by bilateral calcifications in the basal ganglia and other brain regions. The genetic basis of this disorder remains unknown in a significant portion of familial cases. Here, we reported a recessive causal gene, MYORG, for PFBC. Compound heterozygous or homozygous mutations of MYORG co-segregated completely with PFBC in six families, with logarithm of odds (LOD) score of 4.91 at the zero recombination fraction. In mice, Myorg mRNA was expressed specifically in S100β-positive astrocytes, and knockout of Myorg induced the formation of brain calcification at 9 months of age. Our findings provide strong evidence that loss-of-function mutations of MYORG cause brain calcification in humans and mice.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals.

Long non-coding RNAs (lncRNAs) play critical roles in multiple cellular processes in non-small cell lung cancer (NSCLC); however, the involvement of lncRNAs in the transforming growth factor-beta (TGFβ) signaling pathway, the critical tumor cell epithelial-mesenchymal transition (EMT) and metastasis pathway, remains poorly understood. To address this issue, we compared the lncRNAs expression patterns of NSCLC cells treated with and without TGFβ1 treatment. We observed that one of the most prominent hits, TGFβ-induced lncRNA (TBILA), promoted NSCLC progression and was upregulated in tumor tissues. Upregulated TBILA promotes human germinal center-associated lymphoma (HGAL) expression by binding to the Smad transcription factor complex, thereby enhancing RhoA activation. In addition, TBILA induces the S100A7-c-Jun activation domain-binding protein 1 (JAB1) pathway by binding to nuclear S100A7 and enhances pro-survival pathways in NSCLC. These findings have provided us with a new perspective regarding the regulation of the TGFβ signaling pathway in NSCLC and suggest that the lncRNA TBILA can serve as a target for anticancer therapies.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Thrombin is frequently increased in the CNS after injury yet little is known regarding its effects on neural stem cells. Here we show that the subventricular zone (SVZ) of adult mice lacking the high affinity receptor for thrombin, proteinase activated receptor 1 (PAR1), show increased numbers of Sox2+ and Ki-67+ self-renewing neural stem cells (NSCs) and Olig2+ oligodendrocyte progenitors. SVZ NSCs derived from PAR1-knockout mice, or treated with a PAR1 small molecule inhibitor (SCH79797), exhibited enhanced capacity for self-renewal in vitro, including increases in neurosphere formation and BrdU incorporation. PAR1-knockout SVZ monolayer cultures contained more Nestin, NG2+ and Olig2+ cells indicative of enhancements in expansion and differentiation towards the oligodendrocyte lineage. Cultures of NSCs lacking PAR1 also expressed higher levels of myelin basic protein, proteolipid protein and glial fibrillary acidic protein upon differentiation. Complementing these findings, the corpus callosum and anterior commissure of adult PAR1-knockout mice contained greater numbers of Olig2+ progenitors and CC1+ mature oligodendrocytes. Together these findings highlight PAR1 inhibition as a means to expand adult SVZ NSCs and to promote an increased number of mature myelinating oligodendrocytes in vivo that may be of particular benefit in the context of neural injury where PAR1 agonists such as thrombin are deregulated.

Whether fear attenuation is mediated by inhibition of the original memory trace of fear with a new memory trace of safety or by updating of the original fear trace toward safety has been a long-standing question in neuroscience and psychology alike. In particular, which of the two scenarios underlies the attenuation of remote (month-old) fear memories is completely unknown, despite the impetus to better understand this process against the backdrop of enduring traumata. We found-chemogenetically and in an engram-specific manner-that effective remote fear attenuation is accompanied by the reactivation of memory recall-induced neurons in the dentate gyrus and that the continued activity of these neurons is critical for fear reduction. This suggests that the original memory trace of fear actively contributes to remote fear attenuation.

For decades, it has been thought that glutamate and GABA are released by distinct neurons. However, some mouse neurons innervating the lateral habenula (LHb) co-release glutamate and GABA. Here, we mapped the distribution of neurons throughout the rat brain that co-express vesicular transporters for the accumulation of glutamate (VGluT2) or GABA (VGaT) and for GABA synthesis (GAD). We found concentrated groups of neurons that co-express VGluT2, VGaT, and GAD mRNAs within subdivisions of the ventral tegmental area (VTA), entopeduncular (EPN), and supramammillary (SUM) nuclei. Single axon terminals established by VTA, EPN, or SUM neurons form a common synaptic architecture involving asymmetric (putative excitatory) and symmetric (putative inhibitory) synapses. Within the LHb, which receives co-transmitted glutamate and GABA from VTA and EPN, VGluT2 and VGaT are distributed on separate synaptic vesicles. We conclude that single axon terminals from VGluT2 and VGaT co-expressing neurons co-transmit glutamate and GABA from distinct synaptic vesicles at independent synapses.

Abstract

BACKGROUND:

Vertical transmission of Zika virus (ZIKV) leads to infection of neuroprogenitor cells and destruction of brain parenchyma. Recent evidence suggests that the timing of infection as well as host factors may affect vertical transmission. As a result, congenital ZIKV infection may only become clinically apparent in the postnatal period.

OBJECTIVES:

We sought to develop an outbred mouse model of ZIKV vertical transmission to determine if the timing of gestational ZIKV exposure yields phenotypic differences at birth and through adolescence. We hypothesized that later gestational inoculations would only become apparent in adolescence.

METHODS:

To better recapitulate human exposures, timed pregnant Swiss-Webster dams (n=15) were subcutaneously inoculated with 1x104PFU of first passage contemporary ZIKV HN16 strain or a mock injection on embryonic day 4, 8, or 12 with bioactive anti-interferon alpha receptor antibody administered in days preceding and proceeding inoculation. The antibody was given to prevent the robust type I interferon signaling cascade that make mice inherently resistant to ZIKV infection. At birth and adolescence (6 weeks of age) offspring were assessed for growth, brain weight and biparietal head diameters (BPD), and ZIKV viral levels by RT-PCR or in situ hybridization.

RESULTS:

Pups of ZIKV-infected dams infected at e4 and e8 but not e12 were growth restricted (p<0.003). Brain weights were significantly smaller at birth (p=0.01) for e8 ZIKV-exposed offspring. At 6 weeks of age, biparietal diameters (BPD) were smaller for all ZIKV exposed males and females (p<0.05), with e8 exposed males smallest by BPD and growth restriction measurements (weight >2 SD, p=0.0007). All pups and adolescent mice were assessed for ZIKV infection by RT-PCR. Analysis of all underweight pups reveled one to be positive for neuronal ZIKV infection by in situ hybridization, while a second moribund animal was diffusely positive at 8 days of age by ZIKV infectivity throughout the brain, kidneys and intestine.

CONCLUSION:

These findings demonstrate that postnatal effects of infection occurring at single time points continue to be detrimental to offspring in the postnatal period in a subset of littermates and subject to a window of gestational susceptibility coinciding with placentation. This model recapitulates frequently encountered clinical scenarios in non-endemic regions, including the majority of the U.S., where travel related exposure occurs in short and well-defined windows of gestation. Our low rate of infection and relatively rare evidence of congenital Zika syndrome parallels human population-based data.