Daniela Necchi

Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores

We have followed at high resolution the ribosomal protein S6 entering the nucleus of HeLa cells, stopping in some (not all) interchromatin granules clusters and reaching, via Cajal bodies, the nucleolus. There, S6 is assembled with other proteins and rRNA into small ribosomal subunit (SSU), released in the nucleoplasm, and exported through the nuclear pores. We show for the first time the spatial association of nuclear myosin I (NMI) and actin with the SSU already at the nucleolar periphery to the nuclear pore. A blockade of NMI or actin induces an upstream accumulation of the S6 protein en route to the nucleolus, and a temperature lower than normal influences RNA export. Our data strongly suggest a functional relationship of SSU with NMI and actin. In our hypothesis, an active, myosin‐driven movement of the small ribosomal subunit can be responsible for the export of ∼10% of SSUs. This hypothesis is supported by ultrastructural, immunofluorescence, and biochemical analyses. The currently accepted model for the subunit release suggests a diffusive, temperature‐independent mechanism. However, the advantage of the double mechanism would assure that the movement of a part of the subunits could be modulated, increased, or decreased according to the needs of the cell at a specific moment in the cell cycle.—Cisterna, B., Necchi, D., Prosperi, E., Biggiogera, M. Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores. FASEB J. 20, E1257–E1263 (2006)

Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

The cell-cycle inhibitor p21CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.

Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cell nuclear antigen

The cyclin-dependent kinase inhibitor p21CDKN1A plays a fundamental role in the DNA-damage response by inducing cell-cycle arrest, and by inhibiting DNA replication through association with the proliferating cell nuclear antigen (PCNA). However, the role of such an interaction in DNA repair is poorly understood and controversial. Here, we provide evidence that a pool of p21 protein is rapidly recruited to UV-induced DNA-damage sites, where it colocalises with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase δ, XPG and CAF-1. In vivo imaging and confocal fluorescence microscopy analysis of cells coexpressing p21 and PCNA fused to green or red fluorescent protein (p21-GFP, RFP-PCNA), showed a rapid relocation of both proteins at microirradiated nuclear spots, although dynamic measurements suggested that p21-GFP was recruited with slower kinetics. An exogenously expressed p21 mutant protein unable to bind PCNA neither colocalised, nor coimmunoprecipitated with PCNA after UV irradiation. In NER-deficient XP-A fibroblasts, p21 relocation was greatly delayed, concomitantly with that of PCNA. These results indicate that early recruitment of p21 protein to DNA-damage sites is a NER-related process dependent on interaction with PCNA, thus suggesting a direct involvement of p21 in DNA repair.

Regional alterations of the NO/NOS system in the aging brain: a biochemical, histochemical and immunochemical study in the rat

We have used several approaches (immunohistochemistry and enzyme histochemistry, Western blotting, biochemical assay of Ca(2+)-dependent catalytic activity) in order to detect differences in neuronal nitric oxide synthase (nNOS) expression and activity in various brain regions of young-adult (4-month-old) and aged (28-month-old) rats. In most of the brain regions examined (striatum, neocortex, olfactory cortex and hippocampus) some significant decrease in the density per unit area of nNOS neurons, detected either through immunohistochemistry or enzyme histochemistry, was observed in aged rats. However, only in the striatum and olfactory cortex this was accompanied by a significant decrease of the catalytic activity of the constitutive, Ca(2+)-dependent NOS form. In these two regions, the relative level of expression of nNOS protein was also significantly decreased, as assessed by Western blotting of proteic extracts from young-adult and aged rats. Other observed differences were a paler stain of neurons in some brain areas of the aged rats and differences of cellular compartmentalization of the protein in the same rats, as assessed through confocal microscopy. The present observations demonstrate that the expression and activity of nNOS show regionally-specific alterations in the brain of aged healthy rats, with a trend towards decrease, rather than toward increase as suggested by some previous reports. Therefore, hypotheses implicating nitric oxide increase in brain aging should be reconsidered on the basis of a clear-cut distinction between the physiological and the pathological aspects of the aging process.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

Bioactive peptides and serotonin immunocytochemistry in the cerebral ganglia of hibernating Helix aspersa

The role of some neuromodulators and neurotransmitters in the functioning of molluskan cerebral neurons and in their metabolic changes during hibernation has been considered. The cerebral ganglion of mollusks is a center for the integration of different inputs from the sensory areas of the head and for the generation of motor command impulses. During hibernation, animals are deprived of many external sensory stimuli and do not have locomotion and feeding. Immunocytochemistry for bioactive peptides (BAPs), such as SP (Substance P), CCK8 (Cholecystokinin 8/Gastrin), CGRP (Calcitonin-Gene-Related Peptide) and ET (Endothelin), and serotonin was performed on cerebral ganglia of active and hibernating Helix aspersa. The distribution of the immunopositivity was analyzed in different cell-containing areas (procerebrum, mesocerebrum, metacerebrum) and in the neuropiles. With all the antibodies raised against peptides, we observed that only a few neurons, mainly of small and medium size, had immunopositivity during the period of activity, the patterns of distribution being quite similar to those previously described in Helix or other gastropods. Fibers and varicosities with BAP immunopositivity were found in the procerebral and central neuropiles and sometimes around neurons. Serotonin-immunopositive neurons, including the giant neuron, were observed in the metacerebrum; numerous fibers and varicosities immunopositive for serotonin were present in the neuropile areas. In hibernating snails, the number of fibers with BAP and serotonin immunopositivity decreased in several areas of the neuropiles. Moreover, an increased number of neurons of the metacerebrum (two-to four-fold) and mesocerebrum (8- to 28-fold) had BAP-like immunopositivity, and the intensity of the immunoreaction for serotonin of the metacerebral neurons was also higher than in the active snails. These results are discussed, taking into account two hypotheses. The first hypothesis assumes that the increased immunocytochemical staining was really linked to accumulation of BAPs and serotonin. The second hypothesis considers that the antibodies for BAPs recognized a preprotein, the synthesis of BAPs being completed during the active period only. Both the hypotheses account for the co-occurrence and co-localization of two or ore peptides and serotonin and stress that the hibernation condition is of interest for studies on the actual function of single neurons in the cerebral ganglia. Finally, the data are consistent with the changes recently found in other markers of the morphological and functional activity of neurons, demonstrating that the neuromodulation and the neurotransmission are slowed during hibernation.

Nitric oxide‐containing neurons in the nervous ganglia of Helix aspersa during rest and activity: Immunocytochemical and enzyme histochemical detection

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐diaphorase) activity are two cytochemical markers for nitric oxide (NO)‐containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH‐diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS‐containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release. J. Comp. Neurol. 409:274–284, 1999.

Axonal abnormalities in cerebellar Purkinje cells of the Ts65Dn mouse

Ts65Dn mice are a genetic model for Down syndrome. Among others, these mice have cerebellar pathology features which parallel those seen in Down syndrome patients. Both individuals with Down syndrome and Ts65Dn mice have reduced cerebellar volume and numbers of granule and Purkinje cells. In this report, we describe morphological abnormalities of axons of Purkinje cells in the cerebellum of Ts65Dn mice, by using anti-calbindin immunocytochemistry. A consistent number of Purkinje cells shows axons bearing giant varicosities along their transit through the granular layer. The cerebellar arbor vitae made by fasciculated Purkinje cell axons has a patchy appearance, some tracks being devoid of calbindin staining. The infraganglionic plexus, formed by recurrent collaterals of Purkinje cell axons, has enormously increased density, which is evidence for a compensatory reaction to degeneration of distal segments of axons. These alterations are accompanied by strong glial reaction as evidenced by GFAP immunocytochemistry. Moreover, the alterations are more consistent in the anterior lobules of the vermis and intermediate cortex. The axonal pathology of Purkinje cells may explain the impairment in cerebellar functions observed in Ts65Dn mice at the adulthood.

Distribution of calretinin-like immunoreactivity in the brain of Rana esculenta.

The distribution of calretinin-like immunoreactivity has been analyzed in the brain of Rana esculenta. Several neurons of nuclei belonging to sensory pathways, subhabenular area and left habenula were immunopositive. Immunoreactivity was present in fibers of motor and sensory pathways, thalamus, tegmentum and isthmus. The immunolabeling pattern partially overlapped that previously described in the rat. However, in comparison with the rat, fewer cells and fibers were immunoreactive and there were less positive brain nuclei. especially in the pallium, septum and striatum, that were totally negative. Taking into consideration that these regions are rather simple in the frog, the presence of calretinin seems to be consistent with the degree of complexity of brain areas and segregation of different nuclei.

Degradation of p21CDKN1A after DNA damage is independent of type of lesion, and is not required for DNA repair.

The inhibitor of cyclin-dependent kinases p21CDKN1A plays a fundamental role in several pathways involved in the DNA damage response, like checkpoint-mediated cell cycle arrest, transcription, apoptosis, and DNA repair. Although p21 protein level is regulated by proteasomal degradation, the relationship of this process with DNA repair pathways is not yet clear. In addition, the role of protein/protein interaction in regulating turnover of p21 protein, is controversial. Here, we show that in human fibroblasts treated with agents inducing lesions repaired through nucleotide excision repair (NER), or base excision repair (BER), p21 degradation was triggered more by the extent, than by the type of DNA damage, or consequent DNA repair pathway. In fact, lowering the amount of DNA damage resulted in an increased stability of p21 protein. Overexpression of p21 was found to obscure degradation, both for p21wt and a p21 mutant unable to bind PCNA (p21PCNA-). However, when expressed to lower levels, turnover of p21 protein after DNA damage was greatly influenced by interaction with PCNA, since p21PCNA- was more efficiently degraded than wild-type protein. Interestingly, a p21 mutant protein unable to localize in the nucleus because of mutations in the NLS region, was not degraded after DNA damage, thus indicating that nuclear localization is necessary for p21 turnover. Removal of p21 was not required for NER activity, since inhibition of p21 degradation by caffeine did not affect the UV-induced recruitment of repair proteins, such as PCNA and DNA polymerase delta, nor significantly influence DNA repair synthesis, as determined by autoradiography. These results indicate that degradation of p21 is not dependent on a particular DNA repair pathway, and is not required for efficient DNA repair.