An Alternative Splice Variant of Zebrafish Cx52.6 is Expressed in Retinal Horizontal Cells.

Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals.

Complexity of gap junctions between horizontal cells of the carp retina.

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp.

Pannexin1 in the outer retina of the zebrafish, Danio rerio.

In the retina, chemical and electrical synapses couple neurons into functional networks. New candidates encoding for electrical synapse proteins have recently emerged. In the present study, we determined the localization of the candidate protein pannexin1 (zfPanx1) in the zebrafish retina and studied the functional properties of zfPanx1 exogenously expressed in Neuroblastoma 2a (N2a) cells. zfPanx1 was identified on the surface of horizontal cell dendrites invaginating deeply into the cone pedicle near the glutamate release sites of the cones, providing in vivo evidence for hemichannel formation at that location. This strategic position of zfPanx1 in the photoreceptor synapse could potentially allow modulation of cone output. Using whole cell voltage clamp and excised patch recordings of transfected N2a cells, we demonstrated that zfPanx1 forms voltage-activated hemichannels with a large unitary conductance in vitro. These channels can open at physiological membrane potentials. Functional channels were not formed following mutation of a single amino acid within a conserved protein motif recently shown to be N-glycosylated in rodent Panx1. Together, these findings indicate that zfPanx1 displays properties similar to its mammalian homologues and can potentially play an important role in functions of the outer retina.

Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.

Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus.

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unopposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.

Molecular evidence for local denervation of paraspinal muscles in failed-back surgery/postdiscotomy syndrome.

A study was performed to analyze whether local denervation of the medial branch of the dorsal ramus of the lumbar spinal nerve occurs in a patient with postoperative failed-back surgery syndrome/postdiscotomy syndrome (FBSS/PDS). We investigated the effect of the loss of innervation of the multifidus muscle on neuronal nitrite oxide synthetase (n-NOS) and endothelial nitrite oxide synthetase (e-NOS) applying realtime RT-PCR and immunohistochemistry. Our study demonstrates a substantial reduction of n-NOS expression, supporting the view that local denervation of the multifidus is involved in the pathology of FBSS. No regulation of e-NOS was detectable. Interestingly, this change is region-specific and does not occur throughout the entire multifidus segment. This result supports the hypothesis that local denervation of the multifidus muscle is involved in the pathology of FBSS/ PDS.

Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36).

The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.

Developmental regulation and overexpression of the transcription factor AP-2, a potential regulator of the timing of Schwann cell generation.

There is now evidence from in vivo and in vitro studies that the rate of Schwann cell generation is regulated by the balance of two opposing signals, beta neuregulins and endothelins. The beta neuregulins promote the development of precursors to Schwann cells whereas endothelins retard it through an action on endothelin-B receptors. The present work has shown additional controls of this transition, and implicates AP-2 transcription factors, in particular AP-2 alpha, as negative regulators of Schwann cell generation. We found that both AP-2 alpha and AP-2 gamma are present in early embryonic nerves, whereas AP-2 beta was not. Isoform-specific analysis of AP-2 alpha showed that isoform 3 was most abundant with isoforms 1 and 2 present in lesser amounts; isoform 4 was absent. Maximal AP-2 alpha and AP-2 gamma mRNA expression occurred at embryonic day (E) 12/13 in the mouse and at E14/15 in the rat, which correlates with the presence of Schwann cell precursors in the nerve. In both rats and in mice, in vivo and in vitro, downregulation of AP-2 alpha mRNA and protein coincided with one of the main steps in Schwann cell development, the precursor-Schwann cell transition. Moreover, Schwann cell generation was delayed if this downregulation was prevented by enforced expression of AP-2 alpha in precursors. These studies suggest that AP-2 is involved in the control of the timing of Schwann cell development.

Identification of transcriptionally regulated mRNAs from mouse Schwann cell precursors using modified RNA fingerprinting methods.

We have adopted RNA fingerprinting methods to screen for genes that are rapidly up- or down-regulated during normal mammalian development, comparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA template for amplification, the detection of RT-PCR products with a sensitive DNA stain and polyacrylamide gel electrophoresis and rigid selection criteria involving three screening steps are significant improvements on previous methods. Of 19 differentially displayed bands, 15 represented novel genes. The four known cDNA fragments (interleukin enhancer binding factor 1, beta3 subunit of phospholipase C, brain beta-spectrin, and P21 polypeptide) consisted of coding sequences, indicating a high chance of obtaining coding regions. A semiquantitative RT-PCR analysis of three of the four known genes and a cDNA fragment randomly selected from the pool of 15 novel sequences, confirmed that they were regulated between embryo days 12 and 13, as predicted by the display gels. Our results suggest that the combination of methods described here will have wide applicability in studies of other developmental systems where precisely timed changes occur and where only small amounts of RNA can be obtained for analysis.

Studies on the effects of altered PMP22 expression during myelination in vitro.

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot-Marie-Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over- or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT-PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation.

Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth-arrest-specific gene gas3. The biological function of PMP22 is unknown, but recent progress in the analysis of rat Schwann cells expressing altered levels of PMP22 revealed that one role of PMP22 is as a negative growth modulator. We have investigated the influence of rat PMP22 (rPMP22) and a mutant of PMP22 (rPMP22(Tr)) resembling the murine trembler mutation on cell growth of retrovirus-vector-infected mouse NIH3T3 cells. Transduced cells carrying the two different sense constructs expressed rPMP22 and rPMP22(Tr )mRNAs and proteins. Elevated levels of rPMP22 and rPMP22(Tr )significantly reduced fibroblast growth as judged by proliferation assays. Despite a negative modulatory influence of rPMP22 and rPMP22(Tr )on cell proliferation, cell cycle analyses by flow cytometry did not reveal an influence of rPMP22 or rPMP22(Tr )on the synchronous progression of resting NIH3T3 cells from G0 into S phase. However, cell cycle analyses by flow cytometry of asynchronously dividing cultures demonstrated that the expression of rPMP22 and rPMP22(Tr )increased the fraction of cells in the G1 phase of the cell cycle. Furthermore, cell death analyses revealed that, in contrast to control cells and cells carrying the rPMP22(Tr )construct, a significantly increased fraction of NIH3T3 cells expressing rPMP22 exit the proliferation compartment showing hallmarks of programmed cell death. These results indicate that (i) rPMP22 and rPMP22(Tr )act as negative modulators of proliferation in murine fibroblasts probably through extension of the G1 phase of the cell cycle and (ii) rPMP22 but not rPMP22(Tr )promotes programmed death of these cells.

P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non-myelin-forming and myelin-forming Schwann cells, respectively.

We show that in the rat, the major gene of PNS myelin, P0, is expressed long before myelination in the neural crest, Schwann cell precursors, and embryonic Schwann cells irrespective of whether they will myelinate or not. This myelin-independent P0 expression is constitutive and likely to serve as a specific marker for the Schwann cell lineage. The much higher P0 expression accompanying myelination is therefore not new gene expression but strong up-regulation of preexisting basal levels. We provide new evidence that the up-regulation to myelination-related levels depends on positive extrinsic signals and therefore does not represent a constitutive phenotype. P0 mRNA is not detectable in mature non-myelin-forming Schwann cells of the sympathetic trunk, but is detectable after transection, indicating that there is a P0-inhibitory signal associated with mature unmyelinated axons. Thus, the regulation of the P0 gene is complex, encompassing extrinsically signaled amplification superimposed on a highly lineage-specific and constitutive basal expression.

Helix-loop-helix proteins in Schwann cells: a study of regulation and subcellular localization of Ids, REB, and E12/47 during embryonic and postnatal development.

Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells. The mRNA levels were not significantly regulated during development. Nevertheless, by using antibodies against the four different Id proteins, we found clear regulation of some of these genes at the protein level, in particular Id 2, 4, and REB, both in amount and nuclear/cytoplasmic localization. All these proteins are found in the nuclei of Schwann cell precursors but are not seen in nuclei of Schwann cells of newborn nerves. We observed extensive overlap in Id expression, especially in Schwann cell precursors that co-expressed all four Id proteins and REB. We also showed that Id 1 and 2 were up-regulated as Schwann cells progressed through the cell cycle. These data indicate that HLH transcription factors act as regulators of Schwann cell development and point to the existence of as yet unidentified cell type-specific bHLH proteins in these cells.

Full-lenth cloning, expression and cellular localization of rat plasmolipin mRNA, a proteolipid of PNS and CNS.

We have isolated a 1.476 bp cDNA (NTII11) representing a transcript that is differntially expressed during sciatic nerve development and regeneration in the rat. Nucleotide sequence comparison indicates partial identity with a recently isolated plasmolipin cDNA. However, our clone extends the published sequence by 234 bp at the 5' end and predicts a protein that contains an additional 25 amino acids at th N-terminus. The open reading frame of th NTII11 transcript encodes a 19.4 kDa protein with four putative transmembrane domains. Northern blot analyses revealed a tissue-specific expression was confirmed by in situ hybridization, and cellular localization of plasmolipin mRNA was demonstrated in Schwann cells of the sciatic nerve and in glial cells of myelinated brain structures. The steady-state levels of plasmolipin mRNA were markedly altered (i) during development of sciatic nerve and brain. (ii) after sciatic nerve injury, and (ii) in cured Schwann cells maintained under different conditions of cell growth and arrest. Our data indicate a function of plasmolipin during myelination in the central as well as in the peripheral nervous system.

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of two mRNA species indicates a dual function of peripheral myelin protein PMP22 in cell growth and myelination.

Two peripheral myelin protein PMP22 transcripts, CD25 and SR13, have been identified by Northern blot and RNA-polymerase chain reaction (PCR) methods in rat. The CD25 and SR13 mRNA species (each approximately 1.8 kb in size) differ significantly in their 5'-untranslated region (5'-UTR) sequences but encode the same protein. While CD25 mRNA is largely confined to the peripheral nervous system, the SR13 transcript is more ubiquitously expressed in rat tissues. Both transcripts are differentially expressed during postnatal sciatic nerve development. While CD25 expression steadily increases from low levels in neonates up to a maximum at postnatal day 14, SR13 mRNA levels are elevated at birth but decrease throughout adulthood. CD25 and SR13 transcripts are expressed at very low constant levels in developing and adult brain. In degenerating and regenerating segments of injured peripheral nerve changes in CD25 mRNA levels clearly resemble the expression pattern of other myelin genes, whereas expression of SR13 is inversely correlated with the time course of Schwann cell proliferation. In cultured rat meningeal fibroblasts SR13 mRNA expression is strictly growth arrest-specific and independent of forskolin. On the other hand, regulation of CD25 mRNA levels in these cells is more complex with respect to interfering effects of serum and forskolin. In cultured Schwann cells neither CD25 nor SR13 expression is growth arrest-specific. However, both transcript levels are consistently enhanced by forskolin under all conditions of cell growth tested. Expression of CD25 (but not SR13) depends on high Schwann cell density. Our results substantiate the hypothesis that PMP22 serves two biological functions, one related to cell growth (SR13) and another to myelination (CD25).

Deletion of the beta-turn/alpha-helix motif at the exon 2/3 boundary of human c-Myc leads to the loss of its immortalizing function.

The protein product (c-Myc) of the human c-myc proto-oncogene carries a beta-turn/alpha-helix motif at the exon2/exon3 boundary. The amino acid (aa) sequence and secondary structure of this motif are highly conserved among several nuclearly localized oncogene products, c-Myc, N-Myc, c-Fos, SV40 large T and adenovirus (Ad) Ela. Removal of this region from Ad E1a results in the loss of the transforming properties of the virus without destroying its known transregulatory functions. In order to analyse whether deletion of the above-mentioned region from c-Myc has a similar effect on its transformation activity, we constructed a deletion mutant (c-myc delta) lacking the respective aa at the exon2/exon3 boundary. In contrast to the c-myc wild-type gene product, constitutive expression of c-myc delta does not lead to the immortalization of primary mouse embryo fibroblast cells (MEF cells). This result indicates that c-Myc and Ad El a share a common domain which is involved in the transformation process by both oncogenes.