on Journal of neuroscience methods
by Eyman M, Cefaliello C, Bruno AP, Crispino M, Giuditta A
Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction.
on Journal of neuroscience research
by Ferrara E, Cefaliello C, Eyman M, De Stefano R, Giuditta A, Crispino M
We have recently demonstrated that brain plastic events significantly modify synaptic protein synthesis measured by the incorporation of [(35)S]methionine in brain synaptosomal proteins. Notably, in rats learning a two-way active avoidance task, the local synthesis of two synaptic proteins was selectively enhanced. Because this effect may be attributed to transcriptional modulation, we used reverse transcriptase-polymerase chain reaction methods to determine the content of discrete synaptosomal mRNAs in rats exposed to the same training protocol. Correlative analyses between behavioral responses and synaptosomal mRNA content showed that GAT-1 mRNA (a prevalent presynaptic component) correlates with avoidances and escapes in rat cerebellum, while glial fibrillary acid protein mRNA (an astrocytic component) correlates with freezings in cerebellum and cerebral cortex. These observations support the hypothesis that synaptic protein synthesis may be transcriptionally regulated. The cellular origin of synaptic transcripts is briefly discussed, with special regard to those present at large distances from neuron somas.
on Results and problems in cell differentiation
by Crispino M, Cefaliello C, Kaplan B, Giuditta A
The progressive philogenetic lengthening of axonal processes and the increase in complexity of terminal axonal arborizations markedly augmented the demands of the neuronal cytoplasmic mass on somatic gene expression. It is proposed that in an adaptive response to this challenge, novel gene expression functions developed in the axon compartment, consisting of axonal and presynaptic translation systems that rely on the delivery of transcripts synthesized in adjacent glial cells. Such intercellular mode of gene expression would allow more rapid plastic changes to occur in spatially restricted neuronal domains, down to the size of individual synapses. The cell body contribution to local gene expression in well-differentiated neurons remains to be defined. The history of this concept and the experimental evidence supporting its validity are critically discussed in this article. The merit of this perspective lies with the recognition that plasticity events represent a major occurrence in the brain, and that they largely occur at synaptic sites, including presynaptic endings.
on Physiological reviews
by Giuditta A, Chun JT, Eyman M, Cefaliello C, Bruno AP, Crispino M
Neurons have complex and often extensively elongated processes. This unique cell morphology raises the problem of how remote neuronal territories are replenished with proteins. For a long time, axonal and presynaptic proteins were thought to be exclusively synthesized in the cell body, which delivered them to peripheral sites by axoplasmic transport. Despite this early belief, protein has been shown to be synthesized in axons and nerve terminals, substantially alleviating the trophic burden of the perikaryon. This observation raised the question of the cellular origin of the peripheral RNAs involved in protein synthesis. The synthesis of these RNAs was initially attributed to the neuron soma almost by default. However, experimental data and theoretical considerations support the alternative view that axonal and presynaptic RNAs are also transcribed in the flanking glial cells and transferred to the axon domain of mature neurons. Altogether, these data suggest that axons and nerve terminals are served by a distinct gene expression system largely independent of the neuron cell body. Such a local system would allow the neuron periphery to respond promptly to environmental stimuli. This view has the theoretical merit of extending to axons and nerve terminals the marginalized concept of a glial supply of RNA (and protein) to the neuron cell body. Most long-term plastic changes requiring de novo gene expression occur in these domains, notably in presynaptic endings, despite their intrinsic lack of transcriptional capacity. This review enlightens novel perspectives on the biology and pathobiology of the neuron by critically reviewing these issues.
on Rivista di biologia
by Giuditta A, Chun JT, Eyman M, Cefaliello C, Bruno AP, Crispino M
In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.
on Brain research
by Eyman M, Cefaliello C, Ferrara E, De Stefano R, Crispino M, Giuditta A
Synaptosomes from rat brain have long been used to investigate the properties of synaptic protein synthesis. Comparable analyses have now been made in adult male rats trained for a two-way active avoidance task to examine the hypothesis of its direct participation in brain plastic events. Using Ficoll-purified synaptosomes from neocortex, hippocampus and cerebellum, our data indicate that the capacity of synaptosomal protein synthesis and the specific activity of newly synthesized proteins were not different in trained rats in comparison with home-caged control rats. On the other hand, the synthesis of two proteins of 66.5 kDa and 87.6 kDa separated by SDS-PAGE and analyzed by quantitative densitometry was selectively enhanced in trained rats. In addition, the synthesis of the 66.5 kDa protein, but not of the 87.6 kDa protein, correlated with avoidances and escapes and inversely correlated with freezings in the neocortex, while in the cerebellum it correlated with avoidances and escapes. The data demonstrate the participation of synaptic protein synthesis in plastic events of behaving rats, and the selective, region-specific modulation of the synthesis of a synaptic 66.5 kDa protein by the newly acquired avoidance response and by the reprogramming of innate neural circuits subserving escape and freezing responses.
on The European journal of neuroscience
by Eyman M, Cefaliello C, Ferrara E, De Stefano R, Lavina ZS, Crispino M, Squillace A, van Minnen J, Kaplan BB, Giuditta A
The presence of active systems of protein synthesis in axons and nerve endings raises the question of the cellular origin of the corresponding RNAs. Our present experiments demonstrate that, besides a possible derivation from neuronal cell bodies, axoplasmic RNAs originate in periaxonal glial cells and presynaptic RNAs derive from nearby cells, presumably glial cells. Indeed, in perfused squid giant axons, delivery of newly synthesized RNA to the axon perfusate is strongly stimulated by axonal depolarization or agonists of glial glutamate and acetylcholine receptors. Likewise, incubation of squid optic lobe slices with [3H]uridine leads to a marked accumulation of [3H]RNA in the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons. As the cell bodies of these neurons lie outside the optic lobe, the data demonstrate that presynaptic RNA is locally synthesized, presumably by perisynaptic glial cells. Overall, our results support the view that axons and presynaptic regions are endowed with local systems of gene expression which may prove essential for the maintenance and plasticity of these extrasomatic neuronal domains.
on Neuroscience letters
by Eyman M, Crispino M, Kaplan BB, Giuditta A
Nerve endings of squid photoreceptor neurons generate large synaptosomes upon homogenization of the optic lobe. Using several independent methods, these presynaptic structures have been shown to synthesize a wealth of soluble, cytoskeletal and nuclear encoded mitochondrial proteins, and to account for essentially all the translation activity of the synaptosomal fraction. We are now presenting evidence that calexcitin, a learning related, Ca(2+)-binding protein of the B photoreceptors of Hermissenda crassicornis (a mollusk), is synthesized and subjected to post-translational modifications in the squid photoreceptor terminals. In view of the essential role of presynaptic protein synthesis in long-term memory formation in Aplysia, our data suggest that a comparable role may be played by calexcitin synthesized in the squid photoreceptor terminals.
on The Journal of hospital infection
by Crispino M, Boccia MC, Bagattini M, Villari P, Triassi M, Zarrilli R
The aim of this investigation was to study the molecular epidemiology of Stenotrophomonas maltophilia in a university hospital in Italy. Sixty-one clinical isolates were collected from 43 patients during a two-year period. The majority of specimens were from the respiratory tract (41 of 43) of patients in the adult intensive care unit (ICU) (19 of 43) or cystic fibrosis (CF) patients (13 of 43). Genotypic analysis by pulsed-field gel electrophoresis (PFGE) of clinical isolates identified 31 different PFGE patterns. Although most patients were infected or colonized by different S. maltophilia clones, clones with identical genotype were isolated in patients from ICU, where two separate outbreaks were identified. Antimicrobial susceptibility identified a multi-resistant phenotype in all S. maltophilia PFGE clones. The majority of PFGE clones identified (six of seven clones from patients in the ICU) were susceptible to fluoroquinolones. Mechanical ventilation was associated with S. maltophilia acquisition in the ICU.
on Brain research. Molecular brain research
by Crispino M, Capano CP, Aiello A, Iannetti E, Cupello A, Giuditta A
Synaptosomal fractions from rat brain have been analyzed with semi-quantitative RT-PCR methods to determine their content of mRNAs coding for presynaptic, postsynaptic, glial, and neuronal proteins. Each mRNA was determined with reference to the standard HPRT mRNA. In our analyses, mRNAs were considered to be associated with synaptosomes only if their relative amounts were higher than in microsomes prepared in a polysome stabilizing medium, rich in Mg(++) and K(+) ions, or in the homogenate. According to this stringent criterion, the following synaptosomal mRNAs could not be attributed to microsomal contamination and were assumed to derive from the subcellular structures known to harbor their translation products, i.e. GAT-1 mRNAs from presynaptic terminals and glial processes, MAP2 mRNA from dendrites, GFAP mRNA from glial processes, and TAU mRNA from neuronal fragments. This interpretation is in agreement with the involvement of extrasomatic mRNAs in local translation processes.