Gómez-Cuadrado, L;Bullock, E;Mabruk, Z;Zhao, H;Souleimanova, M;Noer, PR;Turnbull, AK;Oxvig, C;Bertos, N;Byron, A;Dixon, JM;Park, M;Haider, S;Natrajan, R;Sims, AH;Brunton, VG;
PMID: 35205651 | DOI: 10.3390/cancers14040904
Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways.
Investigative ophthalmology & visual science
Flood, MD;Wellington, AJ;Eggers, ED;
PMID: 35077550 | DOI: 10.1167/iovs.63.1.33
Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is also growing evidence that retinal dopamine, a neuromodulator that mediates light adaptation, is reduced in early diabetes. Previously, we have shown that after 6 weeks of diabetes, light adaptation is impaired in ON-sustained (ON-s) ganglion cells in the mouse retina. The purpose of this study was to determine whether changes in the response to dopamine receptor activation contribute to this dysfunction.Single-cell retinal patch-clamp recordings from the mouse retina were used to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and 6-week diabetic (STZ-injected) animals. Fluorescence in situ hybridization was also used to assess whether D4R expression was affected by diabetes.D4R activation decreased light-evoked and spontaneous inputs to ON-s ganglion cells in control and diabetic retinas. However, D4R activation caused a smaller reduction in light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as there was no change in D4R mRNA density in the diabetic retinas.These results suggest that the cellular response to dopamine signaling is disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.
Uchiyama, S;Yoshihara, K;Kawanabe, R;Hatada, I;Koga, K;Tsuda, M;
PMID: 34980215 | DOI: 10.1186/s13041-021-00895-3
It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α1A-adrenaline receptors (α1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1aflox/flox mice). A similar reduction was also observed in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α1A-AR antagonist, and by conditional knockout of α1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1aflox/flox mice. Our findings suggest that activation of α1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat.
Development (Cambridge, England)
Marczenke, M;Sunaga-Franze, DY;Popp, O;Althaus, IW;Sauer, S;Mertins, P;Christ, A;Allen, BL;Willnow, TE;
PMID: 34698766 | DOI: 10.1242/dev.200080
Growth arrest-specific 1 (GAS1) acts as a co-receptor to Patched 1 promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in iPSC-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating Notch signaling, essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives Notch pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating Notch and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
Prenatal androgen exposure alters KNDy neurons and their afferent network in a model of polycystic ovarian syndrome
Moore, AM;Lohr, DB;Coolen, LM;Lehman, MN;
PMID: 34346492 | DOI: 10.1210/endocr/bqab158
Polycystic ovarian syndrome (PCOS), the most common endocrinopathy affecting women worldwide, is characterized by elevated luteinizing hormone (LH) pulse frequency due to the impaired suppression of gonadotrophin-releasing hormone (GnRH) release by steroid hormone negative feedback. Although neurons that co-express kisspeptin, neurokinin B and dynorphin (KNDy cells) were recently defined as the GnRH/LH pulse generator, little is understood about their role in the pathogenesis of PCOS. We used a prenatal androgen-treated (PNA) mouse model of PCOS to determine whether changes in KNDy neurons or their afferent network underlie altered negative feedback. First, we identified elevated androgen receptor gene expression in KNDy cells of PNA mice, whereas progesterone receptor and dynorphin gene expression was significantly reduced, suggesting elevated androgens in PCOS disrupt progesterone negative feedback via direct actions upon KNDy cells. Second, we discovered GABAergic and glutamatergic synaptic input to KNDy neurons was reduced in PNA mice. Retrograde monosynaptic tract-tracing revealed a dramatic reduction in input originates from sexually dimorphic afferents in the preoptic area, anteroventral periventricular nucleus, anterior hypothalamic area and lateral hypothalamus. These results reveal two sites of neuronal alterations potentially responsible for defects in negative feedback in PCOS: changes in gene expression within KNDy neurons, and changes in synaptic inputs from steroid hormone-responsive hypothalamic regions. How each of these changes contribute to the neuroendocrine phenotype seen in in PCOS, and the role of specific sets of upstream KNDy afferents in the process, remains to be determined.
Ablation of glucokinase-expressing tanycytes impacts energy balance and increases adiposity in mice
Rohrbach, A;Caron, E;Dali, R;Brunner, M;Pasquettaz, R;Kolotuev, I;Santoni, F;Thorens, B;Langlet, F;
PMID: 34325016 | DOI: 10.1016/j.molmet.2021.101311
Glucokinase (GCK) is critical for glucosensing. In rats, GCK is expressed in hypothalamic tanycytes and appears to play an essential role in feeding behavior. In this study, we investigated the distribution of GCK-expressing tanycytes in mice and their role in the regulation of energy balance.In situ hybridization, reporter gene assay, and immunohistochemistry were used to assess GCK expression along the third ventricle in mice. To evaluate the impact of GCK-expressing tanycytes on arcuate neuron function and mouse physiology, Gck deletion along the ventricle was achieved using loxP/Cre recombinase technology in adult mice.GCK expression was low along the third ventricle but detectable in tanycytes facing the ventromedial arcuate nucleus from bregma -1.5 to -2.2. Gck deletion induced the death of this tanycyte subgroup through the activation of the BAD signaling pathway. The ablation of GCK-expressing tanycytes affected different aspects of energy balance, leading to an increase in adiposity in mice. This phenotype was systematically associated with a defect in NPY neuron function. In contrast, the regulation of glucose homeostasis was mostly preserved, except for glucoprivic responses.This study describes an additional role for GCK in tanycyte biology and highlights the impact of tanycyte loss on the regulation of energy balance.
Spinal astrocyte aldehyde dehydrogenase-2 mediates ethanol metabolism and analgesia in mice
British journal of anaesthesia
Jin, S;Cinar, R;Hu, X;Lin, Y;Luo, G;Lovinger, DM;Zhang, Y;Zhang, L;
PMID: 33934892 | DOI: 10.1016/j.bja.2021.02.035
Little is known about the targets in the CNS that mediate ethanol analgesia. This study explores the role of spinal astrocyte aldehyde dehydrogenase-2 (ALDH2), a key ethanol-metabolising enzyme, in the analgesic effects of ethanol in mice. Astrocyte and hepatocyte ALHD2-deficient mice were generated and tested in acute and chronic pain models. Cell-type-specific distribution of ALDH2 was analysed by RNA in situ hybridisation in spinal slices from astrocytic ALDH2-deficient mice and their wild-type littermates. Spinal ethanol metabolites and γ-aminobutyric acid (GABA) content were measured using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. ALDH2 mRNA was expressed in both astrocytes and neurones in spinal cord slices. Astrocyte ALDH2-deficient mice had decreased expression of ALDH2 mRNA in astrocytes, but not in neurones. Astrocyte ALDH2 deficiency inhibited ethanol-derived acetate, but not acetaldehyde content in spinal cord tissues. Depletion of spinal astrocyte ALDH2 selectively inhibited ethanol-induced anti-nociceptive effect, but not the effect of ethanol, on motor function. Astrocyte ALDH2 deficiency abolished ethanol-induced GABA elevation. The ethanol metabolite acetate produced anti-nociception and increased GABA synthesis in a manner similar to ethanol. I.T. delivery of either GABAA or GABAB receptor antagonists prevented ethanol and acetate-induced analgesia. These findings provide evidence that ALDH2 in spinal astrocytes mediates spinal ethanol metabolism and ethanol-induced analgesic effects by promoting GABA synthesis and GABAergic transmission in spinal cord.
Dvoriantchikova G, Pronin A, Kurtenbach S, Toychiev A, Chou TH, Yee CW, Prindeville B, Tayou J, Porciatti V, Sagdullaev BT, Slepak VZ, Shestopalov VI.
PMID: - | DOI: 10.1038/s41598-018-23894-2
Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.
Frontiers in Neuroscience
Yoo S, Cha D, Kim DW, Hoang TV and Blackshaw S
| DOI: 10.3389/fnins.2019.00240
Leptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.
International journal of molecular sciences
Mayo, F;González-Vinceiro, L;Hiraldo-González, L;Calle-Castillejo, C;Morales-Alvarez, S;Ramírez-Lorca, R;Echevarría, M;
PMID: 36769371 | DOI: 10.3390/ijms24033048
Aquaporin-4 (AQP4) is the most abundant water channel in the central nervous system and plays a fundamental role in maintaining water homeostasis there. In adult mice, AQP4 is located mainly in ependymal cells, in the endfeet of perivascular astrocytes, and in the glia limitans. Meanwhile, its expression, location, and function throughout postnatal development remain largely unknown. Here, the expression of AQP4 mRNA was studied by in situ hybridization and RT-qPCR, and the localization and amount of protein was studied by immunofluorescence and western blotting, both in the brain and spinal cord. For this, wild-type mice of the C57BL/6 line, aged 1, 3, 7, 11, 20, and 60 days, and 18 months were used. The results showed a change in both the expression and location of AQP4 in postnatal development compared to those during adult life. In the early stages of postnatal development it appears in highly myelinated areas, such as the corpus callosum or cerebellum, and as the animal grows, it disappears from these areas, passing through the cortical regions of the forebrain and concentrating around the blood vessels. These findings suggest an unprecedented possible role for AQP4 in the early cell differentiation process, during the first days of life in the newborn animal, which will lead to myelination.
Li, H;Zhao, X;Li, J;Zheng, H;Zhao, Y;Yang, J;Zhou, J;Yang, F;Chen, Y;Zuo, Y;Lai, Q;Long, H;Li, Y;Jin, W;Shi, H;Liu, L;
PMID: 35893674 | DOI: 10.3390/v14081608
Reinfection risk is a great concern with regard to the COVID-19 pandemic because a large proportion of the population has recovered from an initial infection, and previous reports found that primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques without viral presence and pathological injury; however, a high possibility for reinfection at the current stage of the pandemic has been proven. We found the reinfection of SARS-CoV-2 in Syrian hamsters with continuous viral shedding in the upper respiratory tracts and few injuries in the lung, and nasal mucosa was exploited by SARS-CoV-2 for replication and shedding during reinfection; meanwhile, no viral replication or enhanced damage was observed in the lower respiratory tracts. Consistent with the mild phenotype in the reinfection, increases in mRNA levels in cytokines and chemokines in the nasal mucosa but only slight increases in the lung were found. Notably, the high levels of neutralizing antibodies in serum could not prevent reinfection in hamsters but may play roles in benefitting the lung recovery and symptom relief of COVID-19. In summary, Syrian hamsters could be reinfected by SARS-CoV-2 with mild symptoms but with obvious viral shedding and replication, and both convalescent and vaccinated patients should be wary of the transmission and reinfection of SARS-CoV-2.
The Journal of clinical investigation
Ahmadzai, MM;McClain, JL;Dharshika, C;Seguella, L;Giancola, F;De Giorgio, R;Gulbransen, BD;
PMID: 35166239 | DOI: 10.1172/JCI149464
Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.
