Rosalba Parenti

Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons

The connexins are the protein subunits of the gap junction intercellular channels. In the present study a new rat connexin was cloned by degenerate reverse transcription‐polymerase chain reaction and its gene isolated from a mouse genomic library. The nucleotide sequence encodes a protein of 321 amino acids (called Cx36) with highly significant homology to the members of the connexin family. In situ hybridization analysis of rat brain and retina showed the strongest expression in neurons of the inferior olive, the olfactory bulb, the CA3/CA4 hippocampal subfields and several brain‐stem nuclei. An intense expression was also found in the pineal gland and in the retinal ganglion cell and inner nuclear layers. Experiments with neurotoxins, locally injected in the hippocampus or specifically acting on inferior olivary neurons, confirmed the neuronal localization of Cx36. It is the first connexin to be expressed predominantly in mammalian neurons and its identification paves the way for a molecular approach in the study of the role played by gap junctions in the physiology and the pathology of the mammalian brain.

Expression of connexin36 mRNA in adult rodent brain.

&NA; A new member of the connexin gene family, named Connexin36 (Cx36) has, recently, been identified in rodents and shown to be preferentially, if not exclusively, expressed in neurones of the adult CNS. In this study we present a detailed in situ hybridization analysis of the expression pattern of mouse Connexin 36 (mCx36) mRNA in the adult mouse brain, with particular regards to the correlation of mCx36 expression to specific neuronal cell classes and systems. We found that mCx36 was strongly and widely expressed in the brain, including areas where the presence of gap junctions was never detected before. Quantitative analysis of the hybridization signal indicated varying levels of expression in different areas. In particular mCx36 was highly expressed in the neurones at different levels of the motor pathway, the olfactory pathway, the hippocampus, and areas related to the generation of respiratory rhythm. On the contrary, mCx36 was more heterogeneously expressed in nuclei of the sensory pathways. These findings show that mCx36 is the first connexin specifically expressed in neuronal cells in the adult rodent brain. The profiles of expression clearly indicate that mCx36 might play specific roles within different neuronal systems.

Multiple Zonal Projections of the Basilar Pontine Nuclei to the Cerebellar Cortex of the Rat

This study revealed a sagittal zonal pattern of projections to the cerebellar cortex after hydraulic or iontophoretic injections of anterograde tracers (tritiated leucine, wheat germ agglutinin‐horseradish peroxidase, or biotinylated dextrane amine) in the basilar pontine nuclei of Wistar rats. The zonal pattern of projection was observed only after injections of small size, whereas large injections labeled diffusely wide areas of the cerebellar cortex, masking the zonal projection because the fusion of contiguous stripes. Diverging projections to discrete sets of sagittal stripes in the two sides of the cerebellar cortex arose from single injections. The stripes of fiber terminals were sharply delimited on both sides by areas, interstripes, either virtually void of labeling or with a much lower density of labeling. Thus, the areas of the cerebellar cortex were parceled in sets of sagittal compartments, stripes and interstripes, by the pontine projections. Up to five compartments (three stripes and two interstripes) were observed in the paraflocculus, in the copula pyramidis, and in vermal lobule IX. Up to nine compartments (five stripes and four interstripes) were found in the crus I, the lobulus simplex, the paramedian lobule, and vermal lobules VI–VIII. Up to seven compartments (four stripes and three interstripes) were found in the crus II. Single injections into the basilar pontine nuclei usually labeled symmetric areas of the cerebellar cortex, which, in some cases, showed similar number of stripes. When this was not the case, the stripes were usually more numerous in the contralateral than in the ipsilateral side. All areas of the cerebellar cortex were projected upon, with zonation patterns from different regions of the basilar pontine nuclei. The projections of the basilar pontine nuclei to the cerebellar cortex were arranged according to a fixed pattern specific for each cortical area, independently of the number of stripes labeled within. The mean width of the stripes visualized in the single cortical areas of different rats was similar, despite the different size of the injections. The length of the stripes ranged widely in the various areas of different rats. The data collected in this study are consistent with the idea that all the mossy afferents to the cerebellar cortex are arranged with a zonal pattern. J. Comp. Neurol. 430:471–484, 2001.

Expression of pannexin1 in the cns of adult mouse: Cellular localization and effect of 4-aminopyridine-induced seizures

The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.

Cx36 is dynamically expressed during early development of mouse brain and nervous system.

Connexins are structural proteins that are part of the gap junctional channels which couple cells in different tissues. Connexin36 (C×36) is a new member of the connexin gene family, found to be expressed essentially if not exclusively in neuronal cells in adult CNS of mouse, rat and man. Here we have studied C×36 expression during murine embryonic development. C×36 shows a highly dynamic pattern of expression. It is first (E9.5) evident in the forebrain and later its expression expand caudally in the midbrain. At E12.5 its expression correlates with major morphogenetic boundaries in the developing mouse brain, specifically with the dorsoventral telencephalic boundary and the Zona Limitans Intrathalamica. Starting at midgestation (E12.5), it is also expressed in both sympathetic and spinal ganglia, and in two longitudinal stripes along the spinal cord.

A natural antisense transcript against Rad18, specifically expressed in neurons and upregulated during β‐amyloid‐induced apoptosis

Apoptosis, the main form of programmed cell death, is associated to a complex and dynamic transcriptional and post‐transcriptional programme. By microarray analysis, we have previously implicated 241 genes differentially expressed in rat cortical neurons exposed to β‐amyloid (Aβ) protein, the major constituent of amyloid plaques in Alzheimers disease. A large number of identified genes have no name or known function. In the present study, we have investigated one of these genes that encodes for a natural antisense transcript against Rad18 (NAT‐Rad18). Real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) confirmed differential expression of this transcript in cortical neurons exposed to Aβ. In situ hybridization, qRT‐PCR and immunohistochemistry were used to assess the regional and cellular distribution of NAT‐Rad18 in adult rat brain. These experiments showed a widespread distribution of NAT‐Rad18, with the highest levels in the cerebellum, brainstem and cortex, where it was specifically expressed by neurons. NAT‐Rad18 was also strongly expressed in epithelial cells of choroid plexus and cerebral vessels. At the cellular level, expression of Rad18 was counterbalanced by that of its natural antisense transcript, as shown by both in situ hybridization and immunohistochemistry. These experiments suggest the existence of a NAT that exerts a post‐transcriptional control over Rad18.

Pregnancy, embryo-fetal development and nutrition: physiology around fetal programming

Abstract The purpose of this brief narrative review is to highlight the role of nutrition during the gestation period. We focused on the possible effects of imbalance of some nutrients in normal course of pregnancy and embryonic

The pontocerebellar projection: longitudinal zonal distribution of fibers from discrete regions of the pontine nuclei to vermal and parafloccular cortices in the rat

A longitudinal parasagittal organization (alternating labeled and unlabeled stripes) of mossy fiber terminals in the paraflocculus and in the vermal lobule VII of the cerebellum was found after small injections (less than 50 nl) of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into discrete regions of the basilar pontine nuclei (BPN) of rats. Up to three stripes were found within the paraflocculus of both sides, following injections (of about 500 microns in diameter) in either the medial or lateral region of the caudal half of the BPN. Up to five stripes were found in the vermal lobule VII after similar size injections into the rostro-ventral region of the BPN. These results emphasize the possibility that the parasagittal zonal arrangement could be a common pattern of organization shared by climbing and mossy fiber afferents.

Somitogenesis: From somite to skeletal muscle

Myogenesis is controlled by an elaborate system of extrinsic and intrinsic regulatory mechanisms in all development stages. The aim of this review is to provide an overview of the different stages of myogenesis and muscle differentiation in mammals, starting from somitogenesis and analysis of the different portions that constitute the mature somite. Particular attention was paid to regulatory genes, in addition to mesodermal stem cells, which represent the earliest elements of myogenesis. Finally, the crucial role of growth factors, molecules of vital importance in contractile regulation, hormones and their function in skeletal muscle differentiation, growth and metabolism, and the role played by central nervous system, are discussed.

Aberrant expression of TfR1/CD71 in thyroid carcinomas identifies a novel potential diagnostic marker and therapeutic target.

BACKGROUND Type I receptor for transferrin (TfR1/CD71) is overexpressed in several malignant tumors, but no studies are available on thyroid carcinomas. Our previous comparative analyses of the relative distribution of transferrin in benign versus papillary thyroid carcinoma (PTC) tissues highlighted a marked malignancy-associated abundance of the molecule. The aim of the present study was to evaluate whether TfR1/CD71 is also differentially expressed in benign versus malignant thyroid tissues. METHODS Tissue samples, including benign lesions and follicular-derived carcinomas, from 241 patients and a total of 35 benign and malignant fresh specimens were assayed for TfR1/CD71 expression by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. RESULTS We found that transcription of TfR1/CD71 gene is constitutive in thyroid epithelia, but the mRNA is differently translated in benign and malignant tissues. Western blot revealed higher levels of TfR1/CD71 protein in malignant versus benign tissues. Immunohistochemically, most carcinomas exhibited overexpression of the receptor, predominantly in the cytoplasm of neoplastic cells. The highest expression level was detected in primary and metastatic papillary carcinomas and anaplastic carcinomas, with positive results ranging from 86% to 100% of the cases. In contrast, most benign tissues were negative, with only a minority of cases showing focal and weak immunoreactivity. CONCLUSIONS Our findings suggest that altered expression of TfR1/CD71 may be used as a marker helpful in distinguishing PTC from papillary hyperplasia and follicular variant PTC from benign follicular-patterned lesions. Additionally, the present observations support the rationale for the use of radiolabeled transferrin/transferrin analogs and/or anti-TfR1/CD71 antibodies for diagnostic and/or radiotherapeutic purposes in TfR1/CD71-expressing thyroid tumors.