Prenatal Ethanol Exposure and Postnatal Environmental Intervention Alter Dopaminergic Neuron and Microglia Morphology in the Ventral Tegmental Area During Adulthood.

BACKGROUND: Prenatal ethanol exposure (PE) impairs midbrain dopaminergic (DA) neuron function, which might contribute to various cognitive and behavioral deficits, including attention deficits and increased addiction risk, often observed in individuals with fetal alcohol spectrum disorders. Currently, the underlying mechanisms for PE-induced deficits are unclear. PE could lead to neuroinflammation by activating microglia, which play an important role in synaptic function. In the present study, we investigated PE effects on microglial activation and DA neuron density and morphology in the ventral tegmental area (VTA). Since postnatal environmental enrichment can reduce neuroinflammation and ameliorate several PE-induced behavioral deficits, we examined if a postnatal environmental intervention strategy using neonatal handling and postweaning complex housing could reverse PE effects on VTA DA neurons and microglia. METHODS: Pregnant rats received 0 or 6 g/kg/d ethanol by 2 intragastric intubations on gestation days 8 to 20. After birth, rats were reared in the standard laboratory or enriched condition. Male adult rats (8 to 12 weeks old) were used for immunocytochemistry. RESULTS: The results showed that PE decreased VTA DA neuron body size in standardly housed rats. Moreover, there was a significant decrease in numbers of VTA microglial branches and junctions in PE rats, suggesting morphological activation of microglia and possible neuroinflammation. The PE effects on microglia were normalized by postnatal environmental intervention, which also decreased the numbers of microglial branches and junctions in control animals, possibly via reduced stress. CONCLUSIONS: Our findings show an association between PE-induced morphological activation of microglia and impaired DA neuron morphology in the VTA. Importantly, postnatal environmental intervention rescues possible PE-induced microglial activation. These data support that environmental intervention can be effective in ameliorating cognitive and behavioral deficits associated with VTA DA neuron dysfunctions, such as attention deficits and increased addiction risk.

Characterization of iNOS(+) Neutrophil-like ring cell in tumor-bearing mice.

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics.

Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the "functional bit". Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells.

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.

Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel.

Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly accumulate Lucifer Yellow lumenally, it is very likely that processes other than organic anion accumulation are involved in the process of cystogenesis, including the Na,K-ATPase.