Ismail Zaitoun

Lack of sigma-1 receptor exacerbates ALS progression in mice.

The function of the sigma-1 receptor (S1R) has been implicated in modulating the activity of various ion channels. In the CNS S1R is enriched in cholinergic postsynaptic densities in spinal cord motoneurons (MNs). Mutations in S1R have been found in familial cases of amyotrophic lateral sclerosis (ALS). In this study we show that a knockout of S1R in the SOD1*G93A mouse model of ALS significantly reduces longevity (end stage). Electrophysiological experiments demonstrate that MN of mice lacking S1R exhibit increased excitability. Taken together the data suggest the S1R acts as a brake on excitability, an effect that might enhance longevity in an ALS mouse model.

Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle

Genomic imprinting is an epigenetic mechanism that results in monoallelic expression of genes depending on parent-of-origin of the allele. Although the conservation of genomic imprinting among mammalian species has been widely reported for many genes, there is accumulating evidence that some genes escape this conservation. Most known imprinted genes have been identified in the mouse and human, with few imprinted genes reported in cattle. Comparative analysis of genomic imprinting across mammalian species would provide a powerful tool for elucidating the mechanisms regulating the unique expression of imprinted genes. In this study we analyzed the imprinting of 22 genes in human, mouse, and cattle and found that in only 11 was imprinting conserved across the three species. In addition, we analyzed the occurrence of the sequence elements CpG islands, C + G content, tandem repeats, and retrotransposable elements in imprinted and in nonimprinted (control) cattle genes. We found that imprinted genes have a higher G + C content and more CpG islands and tandem repeats. Short interspersed nuclear elements (SINEs) were notably fewer in number in imprinted cattle genes compared to control genes, which is in agreement with previous reports for human and mouse imprinted regions. Long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) were found to be significantly underrepresented in imprinted genes compared to control genes, contrary to reports on human and mouse. Of considerable significance was the finding of highly conserved tandem repeats in nine of the genes imprinted in all three species.

Assessment of genomic imprinting of SLC38A4 , NNAT , NAP1L5 , and H19 in cattle

BackgroundAt present, few imprinted genes have been reported in cattle compared to human and mouse. Comparative expression analysis and imprinting status are powerful tools for investigating the biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. The objective of this study was to assess the imprinting status and pattern of expression of the SLC38A4, NNAT, NAP1L5, and H19 genes in bovine tissues.ResultsA polymorphism-based approach was used to assess the imprinting status of four bovine genes in a total of 75 tissue types obtained from 12 fetuses and their dams. In contrast to mouse Slc38a4, which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle, and we found it expressed in all adult tissues examined. Two single nucleotide polymorphisms (SNPs) were identified in NNAT and used to distinguish between monoallelic and biallelic expression in fetal and adult tissues. The two transcripts of NNAT showed paternal expression like their orthologues in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of tissues, both fetal and adult. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene was paternally expressed in all examined tissues, in contrast to mouse where imprinting is tissue-specific. H19 was found to be maternally expressed like its orthologues in human, sheep, and mouse.ConclusionThis is the first report on the imprinting status of SLC38A4, NAP1L5, and on the expression patterns of the two transcripts of NNAT in cattle. It is of interest that the imprinting of NAP1L5, NNAT, and H19 appears to be conserved between mouse and cow, although the tissue distribution of expression differs. In contrast, the imprinting of SLC38A4 appears to be species-specific.

Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.

Hirschsprungs disease (HSCR) is a congenital condition in which enteric ganglia, formed from neural crest cells (NCC), are absent from the terminal bowel. Dysmotility and constipation are common features of HSCR that persist following surgical intervention. This persistence suggests that the portion of the colon that remains postoperatively is not able to support normal bowel function. To elucidate the defects that underlie this condition, we utilized a murine model of HSCR.

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb−/− mice along extrinsic nerve fibers†

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein‐labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest‐derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest‐derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest‐derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut. J. Comp. Neurol. 520:620–632, 2012.

STAT1-mediated Bim expression promotes the apoptosis of retinal pericytes under high glucose conditions

Hyperglycemia impacts different vascular cell functions and promotes the development and progression of various vasculopathies including diabetic retinopathy. Although the increased rate of apoptosis in pericytes (PCs) has been linked to increased oxidative stress and activation of protein kinase C-δ (PKC-δ) and SHP-1 (Src homology region 2 domain-containing phosphatase-1) tyrosine phosphatase during diabetes, the detailed mechanisms require further elucidation. Here we show that the rate of apoptosis and expression of proapoptotic protein Bim were increased in the retinal PCs of diabetic Akita/+ mice and mouse retinal PCs cultured under high glucose conditions. Increased Bim expression in retinal PCs under high glucose conditions required the sustained activation of signal transducer and activator of transcription 1 (STAT1) through production of inflammatory cytokines. PCs cultured under high glucose conditions also exhibited increased oxidative stress and diminished migration. Inhibition of oxidative stress, PKC-δ or Rho-associated protein kinase I/II was sufficient to protect PCs against apoptosis under high glucose conditions. Furthermore, PCs deficient in Bim expression were protected from high glucose-mediated increased oxidative stress and apoptosis. However, only inhibition of PKC-δ lowered Bim levels. N-acetylcysteine did not affect STAT1 levels, suggesting that oxidative stress is downstream of Bim. PCs cultured under high glucose conditions disrupted capillary morphogenesis of retinal endothelial cells (ECs) in coculture experiments. In addition, conditioned medium prepared from PCs under high glucose conditions attenuated EC migration. Taken together, our results indicate that Bim has a pivotal role in the dysfunction of retinal PCs under high glucose conditions by increasing oxidative stress and death of PCs.

Expression of pigment epithelium‐derived factor and thrombospondin‐1 regulate proliferation and migration of retinal pigment epithelial cells

Age‐related macular degeneration (AMD) is the leading cause of vision loss among elderly. Although the pathogenesis of AMD is associated with retinal pigmented epithelium (RPE) dysfunction and abnormal neovascularization the detailed mechanisms remain unresolved. RPE is a specialized monolayer of epithelial cells with important functions in ocular homeostasis. Pathological RPE damage contributes to major ocular conditions including retinal degeneration and irreversible loss of vision in AMD. RPE cells also assist in the maintenance of the ocular angiogenic balance by production of positive and negative regulatory factors including vascular endothelial growth factor (VEGF), thrombospondin‐1 (TSP1), and pigment epithelium‐derived factor (PEDF). The altered production of PEDF and TSP1, as endogenous inhibitors of angiogenesis and inflammation, by RPE cells have been linked to pathogenesis of AMD and choroidal and retinal neovascularization. However, lack of simple methods for isolation and culture of mouse RPE cells has resulted in limited knowledge regarding the cell autonomous role of TSP1 and PEDF in RPE cell function. Here, we describe a method for routine isolation and propagation of RPE cells from wild‐type, TSP1, and PEDF‐deficient mice, and have investigated their impact on RPE cell function. We showed that expression of TSP1 and PEDF significantly impacted RPE cell proliferation, migration, adhesion, oxidative state, and phagocytic activity with minimal effect on their basal rate of apoptosis. Together, our results indicated that the expression of PEDF and TSP1 by RPE cells play crucial roles not only in regulation of ocular vascular homeostasis but also have significant impact on their cellular function.

Upregulation of imprinted genes in mice: An insight into the intensity of gene expression and the evolution of genomic imprinting

Imprinted genes are expressed monoallelically because one of the two copies is silenced epigentically in a parent-of-origin pattern. This pattern of expression is controlled by differential marking of parental alleles by DNA methylation and chromatin modifications, including both suppressive and permissive histone acetylation and methylation. Suppressive histone modifications mark silenced alleles of imprinted genes, while permissive histone modifications mark the active alleles, suggesting the possibility that imprinted genes would show upregulation in gene expression. However, it is currently unknown whether imprinted genes show such upregulation. To address this question in mice, we estimated the intensity of expression of 59 genes relative to the rest of the genome by analyzing microarray data. Expression levels of 24 genes were validated using quantitative real-time PCR (qPCR). Expression of imprinted genes was found to be upreguled in various adult and embryonic mouse tissues. Consistent with their functions in growth and development, imprinted genes were found to be highly expressed in extraembryonic tissues and progressively upregulated during early embryonic development. In conclusion, upregulation of imprinted genes found in this study is similar to the dosage compensation (twofold upregulation) recently reported for X-linked genes. It has been proposed that the twofold upregulation of X-linked genes has been coupled with low transcriptional variation (noise) which could lead to deleterious effects on the organism. Results of this study suggest a general need for imprinted genes in the mouse to be upregulated to certain levels in order to avoid deleterious effects of variation in gene expression.

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

BackgroundFlow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.FindingsTo address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.ConclusionsWhen considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.

Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1−/−) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1−/− ChEC migration and capillary morphogenesis. TSP1−/− ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1−/− ChEC produced similar levels of VEGF, although TSP1−/− ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.