Eleni Roussa

Transforming Growth Factor β Is Required for Differentiation of Mouse Mesencephalic Progenitors into Dopaminergic Neurons In Vitro and In Vivo: Ectopic Induction in Dorsal Mesencephalon

Tissue engineering is a prerequisite for cell replacement as therapeutic strategy for degenerative diseases, such as Parkinsons disease. In the present study, we investigated regional identity of mesencephalic neural progenitors and characterized their development toward ventral mesencephalic dopaminergic neurons. We show that neural progenitors from ventral and dorsal mouse embryonic day 12 mesencephalon exhibit regional identity in vitro. Treatment of ventral midbrain dissociated neurospheres with transforming growth factor β (TGF‐β) increased the number of Nurr1‐ and tyrosine hydroxylase (TH)‐immunoreactive cells, which can be further increased when the spheres are treated with TGF‐β in combination with sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). TGF‐β differentiation signaling is TGF‐β receptor‐mediated, involving the Smad pathway, as well as the p38 mitogen‐activated protein kinase pathway. In vivo, TGF‐β2/TGF‐β3 double‐knockout mouse embryos revealed significantly reduced numbers of TH labeled cells in ventral mesencephalon but not in locus coeruleus. TH reduction in Tgfβ2−/−/Tgfβ3+/− was higher than in Tgf‐β2+/−/Tgf‐β3−/−. Most importantly, TGF‐β may ectopically induce TH‐immunopositive cells in dorsal mesencephalon in vitro, in a Shh‐ and FGF8‐independent manner. Together, the results clearly demonstrate that TGF‐β2 and TGF‐β3 are essential signals for differentiation of midbrain progenitors toward neuronal fate and dopaminergic phenotype.

Induction and specification of midbrain dopaminergic cells: focus on SHH, FGF8, and TGF-β

Cell-fate decisions along the dorsoventral and anterior–posterior axis of the neural tube are dictated by factors from signaling and organizing centers. According to the prevailing notion, the formation of mesencephalic dopaminergic neurons is directed by diffusable signals from the notochord, floor plate, and isthmic organizer. Sonic hedgehog (Shh), secreted by the notochord and floor plate, and fibroblast growth factor (FGF) 8, secreted by the isthmus, are thought to be key molecules involved in the development of midbrain dopaminergic neurons. During the last decade, the introduction of elegant explant culture systems and the generation of transgenic and mutant mice have greatly contributed to a better understanding of the molecular signals that direct the induction and specification of midbrain dopaminergic neurons. In this context, experimental evidence has challenged the dominant roles of Shh and FGF8 in dopaminergic neuron development. Additional molecules have been identified as being required for the generation of mesencephalic dopaminergic neurons, particularly members of the transforming growth factor beta superfamily.

GDNF promotes neuronal differentiation and dopaminergic development of mouse mesencephalic neurospheres

In the present study we report establishment of a neurosphere culture system derived from mouse ventral mesencephalon at embryonic day 12 and investigate effects of glial cell line-derived neurotrophic factor (GDNF) in the differentiation potential of the neurospheres. The generated neurospheres exhibit stem cell characteristics, i.e. self-renewal capacity and multipotency. Addition of exogenous GDNF resulted in neural differentiation indicated by reduced number of nestin positive cells. GDNF treatment resulted in increased numbers of beta-III-tubulin immunoreactive cells whereas glial fibrillary acidic protein immunoreactivity was not effected. Most importantly, cell numbers expressing early dopaminergic markers, Nurr1 and Ptx3, were significantly higher in GDNF-treated spheres. We conclude that GDNF promotes differentiation of mouse mesencephalic stem cells towards neuronal lineage and most notably dopaminergic development.

Immunolocalization of Anion Exchanger AE2, Na+/H+ Exchangers NHE1 and NHE4, and Vacuolar Type H+-ATPase in Rat Pancreas

We have studied the expression and localization of several H+ and HCO3 – transporters, whose presence in the rat pancreas is still unclear. The Cl–/HCO3 – exchanger AE2, the Na+/H+ exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H+-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at ≈160 kD for AE2, at ≈90 kD and ≈103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl– uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO3 – secretion. The molecular identity of the ductal Cl–/HCO3 – exchanger remains unclear.

Lipocalin-2 (24p3/Neutrophil Gelatinase-associated Lipocalin (NGAL)) Receptor Is Expressed in Distal Nephron and Mediates Protein Endocytosis

Background: Localization and function of the lipocalin-2/NGAL/24p3 receptor (24p3R) in the kidney are unknown. Results: 24p3R is expressed in apical plasma membranes of the distal nephron and mediates high-affinity protein endocytosis in renal cells. Conclusion: 24p3R contributes to protein endocytosis and nephrotoxicity in distal nephron segments. Significance: This is the first study to investigate localization and function of 24p3R in relevant epithelia. In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+7-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+7-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123 ± 50 nm was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+7-MT toxicity in distal nephron segments.

Immunolocalization of anion exchanger AE2 and Na+- HCO 3 − cotransporter in rat parotid and submandibular glands

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Transforming Growth Factor β Cooperates with Persephin for Dopaminergic Phenotype Induction

The aim of the present study was to investigate the putative cooperative effects of transforming growth factor β (TGF‐β) and glial cell line‐derived neurotrophic factor (GDNF) family ligands in the differentiation of midbrain progenitors toward a dopaminergic phenotype. Therefore, a mouse midbrain embryonic day (E) 12 neurospheres culture was used as an experimental model. We show that neurturin and persephin (PSPN), but not GDNF, are capable of transient induction of dopaminergic neurons in vitro. This process, however, requires the presence of endogenous TGF‐β. In contrast, after 8 days in vitro GDNF rescued the TGF‐β neutralization‐dependent loss of the TH‐positive cells. In vivo, at E14.5, no apparent phenotype concerning dopaminergic neurons was observed in Tgf‐β2−/−/gdnf−/− double mutant mice. In vitro, combined TGF‐β/PSPN treatment achieved a yield of approximately 20% TH‐positive cells that were less vulnerable against 1‐methyl‐4‐phenyl pyridinium ion toxicity. The underlying TGF‐β/PSPN differentiation signaling is receptor‐mediated, involving p38 mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase pathways. These results indicate that phenotype induction and survival of fully differentiated neurons are accomplished through distinct pathways and individual factor requirement. TGF‐β is required for the induction of dopaminergic neurons, whereas GDNF is required for regulating and/or maintaining a differentiated neuronal phenotype. Moreover, this study suggests that the combination of TGF‐β with PSPN is a potent inductive cocktail for the generation of dopaminergic neurons that should be considered in tissue engineering and cell replacement therapies for Parkinsons disease.

Distinct expression and subcellular localization patterns of Na+/HCO3- cotransporter (SLC 4A4) variants NBCe1-A and NBCe1-B in mouse brain.

The electrogenic Na+/HCO3- cotransporter (NBCe1) has been identified as a key player for regulation of intracellular pH in several cell types. The present study was undertaken to determine expression and subcellular localization of the NH2-terminal solute carrier (SLC) 4A4 variants NBCe1-A and NBCe1-B in mouse brain using variant-specific antibodies by immunohistochemistry and immunoelectron microscopy. In addition, distribution of NBCe1 variants and activity-dependent regulation was investigated in mouse embryonic day 17.5 (E17.5) hippocampal primary cultures in vitro. The results showed NBCe1-A and NBCe1-B transcript expression in the mouse olfactory bulb, cerebral cortex, hippocampus and cerebellum. NBCe1-A was predominantly expressed in Purkinje cells, granule cells of the dentate gyrus, non-pyramidal cell bodies in cerebral cortex, and in periglomerular and mitral cells in the olfactory bulb. Pyramidal neurons in cerebral cortex and apical cell dendrites in the hippocampus were stained for both NBCe1-A and NBCe1-B. Moreover, NBCe1-B was present in Bergmann glia. At the ultrastructural level, NBCe1-B was preferentially expressed in perivascular astroglial lamellae, whereas both NBCe1 NH2-terminal variants were localized in pre- and postsynaptic compartments. Except for the olfactory bulb, NBCe1-A was always colocalized with calbindin. Treatment of E17.5 primary hippocampal cultures with KCl, showed dramatic downregulation of NBCe1-B mRNA and protein after 60 min, whereas NBCe1-A expression remained unchanged. These data demonstrate for the first time distinct cellular distribution of NBCe1 NH2-terminal variants in mouse brain. NBCe1 may be involved in neuronal modulation, and pH regulation during neuronal activity.

Evidence for KCNQ1 K channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion

Secretion of enzymes and fluid induced by Ca(2+) in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K(+) conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K(+) permeability (IC(50) of approximately 10 microM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E(rev)) of -20.9 +/- 0.9 mV, and a single-channel K(+) conductance (g(K)) of 265.8 +/- 44.0 pS (n = 39). Replacement of cis 500 mM K(+) by 500 mM Na(+) shifted E(rev) to -2.4 +/- 3.6 mV (n = 3), indicating K(+) selectivity. Single-channel analysis identified several K(+) channel groups with distinct channel behaviors. K(+) channels with a g(K) of 651.8 +/- 88.0 pS, E(rev) of -22.9 +/- 2.2 mV, and open probability (P(open)) of 0.43 +/- 0.06 at 0 mV (n = 6) and channels with a g(K) of 155.0 +/- 11.4 pS, E(rev) of -18.3 +/- 1.8 mV, and P(open) of 0.80 +/- 0.03 at 0 mV (n = 3) were inhibited by 100 microM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca(2+)-activated K(+) channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC(50) of approximately 10 microM). Thus KCNQ1 may account for ZG K(+) conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.

Anatomic characteristics of the furcation and root surfaces of molar teeth and their significance in the clinical management of marginal periodontitis.

Treatment of periodontitis which involves the furcation area of molar teeth has always been a challenge. Sixty extracted human molar teeth were used for an anatomic study to clarify the possible morphologic factors which may affect the diagnosis, prognosis, and clinical management of periodontally involved teeth. Teeth were embedded in methylmethacrylate and cut in 70 μm cross sections from the cementoenamel junction to the apex. Cervical enamel projection, root and root trunk lengths, root proximities as well as depths of root concavities coronally, apically, and at the furcation roof were measured. Cervical enamel projections were observed in 30% of the teeth examined. The shortest root trunk was found at the buccal aspect of mandibular first molars, permitting furcation involvement at early stages of periodontal disease. The most apically located furcation was found at the distal site of maxillary first molars. Furcation roofs showed severe concavities and complex cementum morphology. Teeth exhibiting cervical enamel projection were found to have deeper root concavities compared to teeth without cervical enamel projection and mesial roots of mandibular molars possessed greater furcal concavities than distal roots. The presence of root concavities complicates the diagnosis of furcation involvement and restricts access of periodontal instruments, resulting in incomplete treatment. Furcation anatomy may also influence the long‐term prognosis of the teeth by favoring the retention of bacterial deposits and making oral hygiene procedures almost impossible. The present study shows that knowledge of a tooths unique anatomic characteristics is a prerequisite for effective periodontal therapy. Clin. Anat. 11:177–186, 1998.