Sheath Cell Invasion and Trans-differentiation Repair Mechanical Damage Caused by Loss of Caveolae in the Zebrafish Notochord.

The notochord, a conserved axial structure required for embryonic axis elongation and spine development, consists of giant vacuolated cells surrounded by an epithelial sheath [1-3]. During morphogenesis, vacuolated cells maintain their structural integrity despite being under constant mechanical stress [4]. We hypothesized that the high density of caveolae present in vacuolated cells [5, 6] could buffer mechanical tension. Caveolae are 50- to 80-nm membrane invaginations lined by cage-like polygonal structures [7, 8] formed by caveolin 1 (Cav1) or Cav3 and one of the cavin proteins [6, 9-11]. Recent in vitro work has shown that plasma membrane caveolae constitute a membrane reservoir that can buffer mechanical stresses such as stretching or osmotic swelling [12]. Moreover, mechanical integrity of vascular and muscle cells is partly dependent on caveolae [13-15]. However, the in vivo mechano-protective roles of caveolae have only begun to be explored. Using zebrafish mutants for cav1, cav3, and cavin1b, we show that caveolae are essential for notochord integrity. Upon loss of caveola function, vacuolated cells collapse at discrete positions under the mechanical strain of locomotion. Then, sheath cells invade the inner notochord and differentiate into vacuolated cells, thereby restoring notochord function and allowing normal spine development. Our data further indicate that nucleotides released by dying vacuolated cells promote sheath cell vacuolization and trans-differentiation. This work reveals a novel structural role for caveolae in vertebrates and provides unique insights into the mechanisms that safeguard notochord and spine development.

Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish.

Skeletal muscles provide metazoans with the ability to feed, reproduce and avoid predators. In humans, a heterogeneous group of genetic diseases, termed muscular dystrophies (MD), lead to skeletal muscle dysfunction. Mutations in the gene encoding Caveolin-3, a principal component of the membrane micro-domains known as caveolae, cause defects in muscle maintenance and function; however it remains unclear how caveolae dysfunction underlies MD pathology. The Cavin family of caveolar proteins can form membrane remodeling oligomers and thus may also impact skeletal muscle function. Changes in the distribution and function of Cavin4/Murc, which is predominantly expressed in striated muscles, have been reported to alter caveolae structure through interaction with Caveolin-3. Here, we report the generation and phenotypic analysis of murcb mutant zebrafish, which display impaired swimming capacity, skeletal muscle fibrosis and T-tubule abnormalities during development. To understand the mechanistic importance of Murc loss of function, we assessed Caveolin-1 and 3 localization and found it to be abnormal. We further identified an in vivo function for Murc in Erk signaling. These data link Murc with developmental defects in T-tubule formation and progressive muscle dysfunction, thereby providing a new candidate for the etiology of muscular dystrophy.

Pleiotropic functions of pituitary adenylyl cyclase-activating polypeptide on retinal ontogenesis: involvement of KLF4 in the control of progenitor cell proliferation.

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.

AT1 and aldosterone receptors blockade prevents the chronic effect of nandrolone on the exercise-induced cardioprotection in perfused rat heart subjected to ischemia and reperfusion.

PURPOSE: Myocardial tolerance to ischaemia/reperfusion (I/R) injury is improved by exercise training, but this cardioprotection is impaired by the chronic use of anabolic androgenic steroids (AAS). The present study evaluated whether blockade of angiotensin II receptor (AT1-R) with losartan and aldosterone receptor (mineralocorticoid receptor, MR) with spironolactone could prevent the deleterious effect of AAS on the exercise-induced cardioprotection. METHODS AND RESULTS: Male Wistar rats were exercised and treated with either vehicle, nandrolone decanoate (10 mg/kg/week i.m.) or the same dose of nandrolone plus losartan or spironolactone (20 mg/kg/day orally) for 8 weeks. Langendorff-perfused hearts were subjected to I/R and evaluated for the postischaemic recovery of left ventricle (LV) function and infarct size. mRNA and protein expression of angiotensin II type 1 receptor (AT1-R), mineralocorticoid receptor (MR), and KATP channels were determined by reverse-transcriptase polymerase chain reaction and Western blotting. Postischaemic recovery of LV function was better and infarct size was smaller in the exercised rat hearts than in the sedentary rat hearts. Nandrolone impaired the exercise-induced cardioprotection, but this effect was prevented by losartan (AT1-R antagonist) and spironolactone (MR antagonist) treatments. Myocardial AT1-R and MR expression levels were increased, and the expression of the KATP channel subunits SUR2a and Kir6.1 was decreased and Kir6.2 increased in the nandrolone-treated rat hearts. The nandrolone-induced changes of AT1-R, MR, and KATP subunits expression was normalized by the losartan and spironolactone treatments. CONCLUSION: The chronic nandrolone treatment impairs the exercise-induced cardioprotection against ischaemia/reperfusion injury by activating the cardiac renin-angiotensin-aldosterone system and downregulating KATP channel expression.

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

BACKGROUND: Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. METHODOLOGY/PRINCIPAL FINDINGS: We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (ß-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. CONCLUSION: Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Pituitary adenylyl cyclase-activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1.

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase-activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP-cAMP-dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP-specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [(3)H]thymidine as well as the number of 5-bromo-2'-deoxyuridine-positive and cyclin D1-positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP-PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell-extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.

Hop/STI1 modulates retinal proliferation and cell death independent of PrPC.

Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP(C)). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP(C) dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 (alpha-STI1) blocked both ganglion cell and NBL cell death independent of PrP(C). cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while alpha-STI1 increased proliferation in the developing retina, both independent of PrP(C). We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP(C).

Glial-derived neurotrophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell death by both PI3K/AKT and MEK/ERK signaling pathways.

We investigated the neuroprotective effect of glial-derived neurotrophic factor (GDNF) upon alcohol-exposed B92 cultures, as well as the role of the cytoskeleton and mitogen-activated protein kinase (MAPK) pathways in this effect. Ethanol (EtOH) was added to cultures, either alone or in combination with 30 ng/ml GDNF. Exposure to EtOH (86 and 172 mM; 60 and 120 min) increased the frequency of apoptotic cells identified by nuclear DNA staining with 4,6-diamidino-2-phenylindole (DAPI). Cultures treated with GDNF showed a decrease in ethanol-induced apoptosis. A jun N-terminal kinase (JNK) pathway is activated by EtOH and their pharmacological inhibition (by SP600125) neutralized ethanol-induced apoptosis, suggesting a role for JNK in EtOH neurotoxicity. Immunocytochemically detected phospho-JNK (p-JNK) showed an unusual filamental expression, and localized together with actin stress fibers. Examination of the cytoskeleton showed that EtOH depolymerized actin filaments, inducing p-JNK dissociation and translocation to the nucleus, which suggests that released p-JNK may contribute to glial cell death after EtOH exposure. Treatment with GDNF, in turn, may neutralize the ethanol-induced cell death pathway. Either a phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitor (LY294002) or an inhibitor of the extracellular signal-regulated kinase (ERK) 1, 2 pathways (UO126) failed to neutralize GDNF protective effects. However, the simultaneous use of both inhibitors blocked the protective effect of GDNF, suggesting a role for both signaling cascades in the GDNF protection. These findings provide further insight into the mechanism involved in ethanol-induced apoptosis and the neurotrophic protection of glial cells.

Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil.

Bidens pilosa (Asteraceae), a medicinal plant used worldwide, has antimalarial activity as shown in previous work. This study tested ethanol extracts from wild plants collected in three different regions of Brazil and from plants cultivated in various soil conditions. The extracts were active in mice infected with P. berghei: doses of < or =500 mg/kg administered by oral route reduced malaria parasitaemia and mouse mortality; higher doses were found to be less effective. Tested in vitro against three P. falciparum isolates, two chloroquine resistant and one mefloquine resistant, the plants cultivated under standard conditions, and in humus enriched soil, were active; but the wild plants were the most active. Analysis using thin layer chromatography demonstrated the presence of flavonoids (compounds considered responsible for the antimalarial activity) in all plants tested, even though at different profiles. Because B. pilosa is proven to be active against P. falciparum drug-resistant parasites in vitro, and in rodent malaria in vivo, it is a good candidate for pre-clinical tests as a phytotherapeutic agent or for chemical isolation of the active compounds with the aim of finding new antimalarial drugs.

Accidental infection of laboratory worker with vaccinia virus.

We report the accidental needlestick inoculation of a laboratory worker with vaccinia virus. Although the patient had previously been vaccinated against smallpox, severe lesions appeared on the fingers. Western blot and polymerase chain reaction-restriction fragment length polymorphism were used to analyze the virus recovered from the lesions. The vaccinia virus-specific immunoglobulin G levels were measured by enzyme-linked immunosorbent assay. Our study supports the need for vaccination for laboratory workers that routinely handle orthopoxvirus.