Viktoria Denes

Effects of pituitary adenylate cyclase activating polypeptide in retinal degeneration induced by monosodium-glutamate.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with a wide range of effects in the central and peripheral nervous systems. PACAP has well-documented neurotrophic and neuroprotective actions in both in vitro and in vivo models of different neuronal injuries. The aim of the present study was to investigate the possible neuroprotective effect of PACAP in retinal degeneration induced by monosodium-glutamate (MSG) in neonatal rats. Preceding the MSG treatment, PACAP (1 or 100pmol/5mul) was injected unilaterally into the vitreous body on postnatal days 1, 5 and 9. Immediately after the PACAP treatment, pups were treated with 2mg/g body weight MSG subcutaneously. At 3 weeks of age, rats were sacrificed and retinas were removed and processed for histological examination. Our results show that MSG treatment caused severe degeneration, primarily of the inner retinal layers. The thickness of the entire retina was only approximately half of that of the normal retinas, and the inner nuclear layer seemed to be fused with the ganglionic cell layer, with no discernible inner plexiform layer. Retinas of animals treated with 1pmol PACAP showed a similar degree of degeneration. However, retinas of rats treated with 100pmol PACAP showed significantly less damage, with clearly distinguishable inner retinal layers. In summary, our present study shows that local PACAP treatment could attenuate the retinal degeneration induced by the excitotoxic effects of glutamate.

Laminin deficits induce alterations in the development of dopaminergic neurons in the mouse retina

Genetically modified mice lacking the beta2 laminin chain (beta2null), the gamma3 laminin chain (gamma3 null), or both beta2/gamma3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in gamma3 null mice. In beta2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Müller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Müller glial cell and/or the retinal ganglion cell.

High male chimerism in the female breast shows quantitative links with cancer

Clinical observations suggest that pregnancy provides protection against cancer. The mechanisms involved, however, remain unclear. Fetal cells are known to enter the mothers circulation during pregnancy and establish microchimerism. We investigated if pregnancy‐related embryonic/fetal stem cell integration plays a role in breast cancer. A high‐sensitivity Y‐chromosome assay was developed to trace male allogeneic cells (from male fetus) in females. Fixed‐embedded samples (n = 206) from both normal and breast cancer patients were screened for microchimerism. The results were combined with matching clinicopathological and histological parameters and processed statistically. The results show that in our samples (182 informative) more than half of healthy women (56%) carried male cells in their breast tissue for decades (n = 68), while only one out of five in the cancer sample pool (21%) (n = 114) (odds ratio = 4.75, CI at 95% 2.34–9.69; p = 0.0001). The data support the notion that a biological link may exist between chimerism and tissue‐integrity. The correlation, however, is non‐linear, since male microchimerism in excess (“hyperchimerism”) is also involved in cancer. The data suggest a link between hyperchimerism and HER2‐type cancers, while decreased chimerism (“hypochimerism”) associates with ER/PR‐positive (luminal‐type) breast cancers. Chimerism levels that correlate with protection appear to be non‐random and share densities with the mammary progenitor components of the stem cell lineage in the breast. The results suggest that protection may involve stem/progenitor level interactions and implicate novel quantitative mechanisms in chimerism biology.

Neurochemical characterization of nervous elements innervating the body wall of earthworms ( Lumbricus , Eisenia ): immunohistochemical and pharmacological studies

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological–physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus submuscularis is composed of nerve fibres only, whereas the plexus subepithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT3 and GABAB receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.

Metastasis blood test by flow cytometry: In vivo cancer spheroids and the role of hypoxia

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis—compared to the high circulating tumor‐cell numbers (CTCs)—appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid‐size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light‐scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC‐association with in vivo spheroids. We propose that the mechanism involves a portable “micro‐niche” in spheroids that may support CSC‐survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression.

Loss of a cohesin-linked suppressor APRIN (Pds5b) disrupts stem cell programs in embryonal carcinoma: an emerging cohesin role in tumor suppression.

Cohesins appear to have critical functions beyond mitotic cohesion. Our data on a cohesin-associated Pds5-paralog, APRIN, indicate a novel cohesin role in stem cell differentiation. APRIN/Pds5B is lost in many cancers and it is a putative tumor suppressor. Its mutations in the germ line, however, generate birth defects. We reasoned that as both cancer and birth defects share disrupted stem cell differentiation, the data suggest an APRIN/Pds5B cohesin function in stem cells. We used an embryonal carcinoma stem cell model and show here that (i) APRIN expression is precisely coordinated with stem cell differentiation; (ii) this coordination involves surface-contact and endocrine pathways; and (iii) APRIN/Pds5b coordination is critical in stem/progenitor exit decisions. APRIN knockdown disrupted Oct4, Nanog and SOX2 patterns, differentiation failed and the resulting immature proliferative cells did not progress beyond proneural progenitor phase. Furthermore, the phenotype—blocked progenitor exit (Mash-1+); failed E-cadherin exit (E-Cadhlow+); incomplete N-cadherin transition (N-Cadhlow+); retained proliferative capacity (c-myc+); irregular stemness (SOX2late++) and lost response to contact and hormonal cues—shares similarities with cancer-initiating cells. The data suggest novel APRIN/Pds5B-linked cohesin roles in stem/progenitor programs and a new mechanism in tumor suppression.

APRIN is a unique Pds5 paralog with features of a chromatin regulator in hormonal differentiation

Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in the Pds5 family). Our results suggest that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. The results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.

Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern.

Abstract Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm2 and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis.

PAC1-expressing structures of neural retina alter their PAC1 isoform splicing during postnatal development

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family, exerts various effects on neuronal development as mediated by the differential expression of PAC1 receptor (PAC1-R) isoforms. The expression changes of PAC1-R isoforms (Hip, Hop1) reported in correlation with retinal development suggest an isoform switch during the second postnatal week. Our aim is to determine the exact period of the isoform shift and to describe the PAC1-R-immunoreactive structures appearing from postnatal day 5 (P5) to P10 in the rat retina. The ratio of Hip and Hop1 receptors was assessed and changes in their expression were followed by Taqman and SybrGreen-based quantitative polymerase chain reaction. For the detection of PAC1-R-expressing retinal structures, anti-PAC1-R, anti-calbindin, anti-protein kinase C, anti-glutamine synthetase, anti-HPC1 and anti-Brn3a antibodies were utilized. At the transcript level, a marked decrease to an undetectable level was measured in Hip mRNA expression from P6 to P9. Hop1 expression appeared to be unchanged from P6 to P9, followed by a significant elevation at P10. A Hip/Hop1 isoform shift occurred between P6 and P7. Immunostaining showed strong PAC1-R labeling from P5 to P10 in ganglion, amacrine, horizontal and rod bipolar neurons and in glial Muller cell processes. The Hop1 isoform was predominantly expressed in various types of retinal cell beginning at P7, because of a dramatic reduction in Hip mRNA level. As the Hop1 receptor is coupled to different signaling cascades, this isoform shift might alter the physiological role of PACAP during this particular period.

Accelerated retinal aging in PACAP knock-out mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.