Brain synaptosomes harbor more than one cytoplasmic system of protein synthesis.

Synaptosomal protein synthesis from rat brain is selectively increased by learning and is massively enhanced during the recovery period from brain ischemia. To lay the groundwork for identification of the involved synaptic elements, we examined the effects induced by varying the concentrations of extracellular cations and endogenous calcium. Most of the recorded rate response curves exhibited biphasic profiles that suggested the presence of more than one translation system. Because comparable profiles were obtained by fully inhibiting mitochondrial translation, the data indicated the involvement of cytoplasmic translation systems present in different synaptosomal classes. Their properties may be individually investigated by exploiting the partially inhibited conditions we have described. The identification of the synaptic elements from which they originated and their newly synthesized proteins will significantly expand our understanding of the synaptic contribution to brain plastic events.

Training old rats selectively modulates synaptosomal protein synthesis.

We have previously shown that the local synthesis of two synaptic proteins of 66.5-kDa and 87.6-kDa is selectively enhanced in male adult rats trained for a two-way active avoidance task. We report here that a comparable but not identical response occurs in 2-year-old male rats trained for the same task. In the latter age group, the local synthesis of the 66.5-kDa protein markedly increases in cerebral cortex, brainstem, and cerebellum, with a somewhat lower increment in synthesis of the 87.6-kDa protein. On the other hand, the newly synthesized 87.6-kDa protein correlates with avoidances and escapes and inversely correlates with freezings in cerebral cortex and brainstem, whereas the correlations of the newly synthesized 66.5-kDa protein remain below significance. These correlative patterns are sharply at variance with those present in trained adult rats. Our data confirm that the local system of synaptic protein synthesis is selectively modulated by training and show that the synaptic response of old rats differs from that of adult rats as reflected in behavioral responses.

Brain Metabolic DNA: A Long Story and Some Conclusions.

We have previously outlined the main properties of brain metabolic DNA (BMD) and its involvement in circadian oscillations, learning, and post-trial sleep. The presence of BMD in certain subcellular fractions and their behavior in cesium gradients have suggested that BMD originates from cytoplasmic reverse transcription and subsequently acquires a double-stranded configuration. More recently, it has been reported that some DNA sequences of cytoplasmic BMD in learning mice are different from that of the control animals. Furthermore, BMD is located in vicinity of the genes involved in different modifications of synaptic activity, suggesting that BMD may contribute to the brain's response to the changing environment. The present review outlines recent data with a special emphasis on reverse transcription of BMD that may recapitulate the molecular events at the time of the "RNA world" by activating mitochondrial telomerase and generating RNA templates from mitochondrial transcripts. The latter unexpected role of mitochondria is likely to promote a better understanding of mitochondrial contribution to cellular interactions and eukaryotic evolution. An initial step regards the role of human mitochondria in embryonic BMD synthesis, which is exclusively of maternal origin. In addition, mitochondrial transcripts involved in reverse transcription of BMD might possibly reveal unexpected features elucidating mitochondrial involvement in cancer events and neurodegenerative disorders.

Sequences of Reverse Transcribed Brain DNA Are Modified by Learning.

Brain metabolic DNA (BMD) is continuously synthesized by reverse transcription in presynaptic synaptosomes and astroglia, and is partly transferred to nuclei after acquiring the double stranded configuration. Synthesis and turnover of BMD are markedly dependent on brain activity, as shown by circadian oscillations, environmental enrichment and impoverishment, and a variety of learning protocols. In rodents learning a two-way active avoidance task, BMD synthesis doubles, thus raising the possibility that sequences of learning BMD may differ from control BMD. The hypothesis has now been examined by sequencing cytoplasmic BMD. The present data indicate that most high-quality mapped BMD fragments hosting more than seven sequences are present in all mice. Three of them are exclusively present in learning BMD and four in control BMD. In addition, the annotated genes closest to them are mostly involved in modulating synaptic activity. The data support the conclusion that learning BMD sequences encode brain responses to the modified environment.

Brain metabolic DNA: recent evidence for a mitochondrial connection.

This review highlights recent data concerning the synthesis of brain metabolic DNA (BMD) by cytoplasmic reverse transcription and the prompt acquisition of the double-stranded configuration that allows its partial transfer to nuclei. BMD prevails in the mitochondrial fraction and is present in presynaptic regions and astroglial processes where it undergoes a turnover lasting a few weeks. Additional data demonstrate that BMD sequences are modified by learning, thus indicating that the modified synaptic activity allowing proper brain responses is encoded in learning BMD. In addition, several converging observations regarding the origin of BMD strongly suggest that BMD is reverse transcribed by mitochondrial telomerase.

Deregulated Local Protein Synthesis in the Brain Synaptosomes of a Mouse Model for Alzheimer's Disease.

While protein synthesis in neurons is largely attributed to cell body and dendrites, the capability of synaptic regions to synthesize new proteins independently of the cell body has been widely demonstrated as an advantageous mechanism subserving synaptic plasticity. Thus, the contribution that local protein synthesis at synapses makes to physiology and pathology of brain plasticity may be more prevalent than initially thought. In this study, we tested if local protein synthesis at synapses is deregulated in the brains of TgCRND8 mice, an animal model for Alzheimer's disease (AD) overexpressing mutant human amyloid precursor protein (APP). To this end, we used synaptosomes as a model system to study the functionality of the synaptic regions in mouse brains. Our results showed that, while TgCRND8 mice exhibit early signs of brain inflammation and deficits in learning, the electrophoretic profile of newly synthesized proteins in their synaptosomes was subtly different from that of the control mice. Interestingly, APP itself was, in part, locally synthesized in the synaptosomes, underscoring the potential importance of local translation at synapses. More importantly, after the contextual fear conditioning, de novo synthesis of some individual proteins was significantly enhanced in the synaptosomes of control animals, but the TgCRND8 mice failed to display such synaptic modulation by training. Taken together, our results demonstrate that synaptic synthesis of proteins is impaired in the brain of a mouse model for AD, and raise the possibility that this deregulation may contribute to the early progression of the pathology.

Synaptic mRNAs are modulated by learning.

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.

DNA in Squid Synaptosomes.

The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.

Brain Metabolic DNA Is Reverse Transcribed in Cytoplasm: Evidence by Immunofluorescence Analysis.

In a previous study (Mol Neurobiol 55:7476-7486, 2017), newly synthesized brain metabolic DNA (BMD) from rat subcellular fractions has been shown to behave as a DNA-RNA hybrid when analyzed in cesium gradients at early [3H] thymidine incorporation times but to assume the double-stranded configuration at later times. Conversely, BMD from purified nuclei displayed the dsDNA configuration even at early incorporation times. The results were interpreted to support the BMD origin by reverse transcription in the cytoplasm and its later acquisition of the double-stranded configuration before the partial transfer to the nuclei. This interpretation has now been confirmed by immunofluorescence analyses of newly synthesized BrdU-labeled BMD from the mouse brain that demonstrates its cytoplasmic localization and colocalization with DNA-RNA hybrids. In addition, BrdU-labeled BMD has been shown to colocalize with astroglial anti-GFAP antibodies and with presynaptic anti-synaptophysin antibodies.

Natural computing and biological evolution: a new paradigm.

After a brief outline of the available hypotheses on the mechanism of biological evolution, attention is called on the global nature of the variations leading to the generation of new species. Integrated changes may hardly be attributed to beneficial random mutations of single traits even if assisted by a phylogenetic elimination of poorly adapted individuals. Rather, integrated variations are likely to reflect the outcome of cybernetic algorithms (natural computing) operating on organism's resources and impending environmental changes. As all organisms are endowed with computing capacities that modulate and integrate ontogenetic development and maintenance of biological functions, structures, and behaviors, these capacities are assumed to have moulded the evolutionary variations of organisms, and their transfer to the progeny.

Brain Metabolic DNA in Rat Cytoplasm.

Brain metabolic DNA (BMD) is not involved in cell division or DNA repair but is modulated by memory acquisition, sleep processing, and circadian oscillations. Using routine methods of subcellular fractionation, newly synthesized BMD from male rats is shown to be localized in crude nuclear, mitochondrial, and microsomal fractions and in two fractions of purified nuclei. Sub-fractionation of the mitochondrial fraction indicates the prevalent localization of BMD in free mitochondria and to a lesser degree in synaptosomes and myelin. Cesium density profiles of homogenate, subcellular fractions, and purified nuclei obtained after incorporation periods from 30 min to 4 h indicate that BMD synthesis takes place by reverse transcription in cytoplasmic organelles. Following the acquisition of the double-stranded structure, BMD is transferred to nuclei. Kinetic analyses lasting several weeks highlight the massive BMD turnover in subcellular fractions and purified nuclei and its dependence on age. Data are in agreement with the role of BMD as a temporary information store of cell responses of potential use in comparable forthcoming experiences.

Squid Giant Axons Synthesize NF Proteins.

Squid giant axon has been an excellent model system for studying fundamental topics in neurobiology such as neuronal signaling. It has been also useful in addressing the questions of local protein synthesis in the axons. Incubation of isolated squid giant axons with [35S]methionine followed by immunoprecipitation with a rabbit antibody against all squid neurofilament (NF) proteins demonstrates the local synthesis of a major 180 kDa NF protein and of several NF proteins of lower molecular weights. Their identification as NF proteins is based on their absence in the preimmune precipitates. Immunoprecipitates washed with more stringent buffers confirmed these results. Our data are at variance with a recent study based on the same experimental procedure that failed to visualize the local synthesis of NF proteins by the giant axon and thereby suggested their exclusive derivation from nerve cell bodies (as reported by Gainer et al. in Cell Mol Neurobiol 37:475-486, 2017). By reviewing the pertinent literature, we confute the claims that mRNA translation is absent in mature axons because of a putative translation block and that most proteins of mature axons are synthesized in the surrounding glial cells. Given the intrinsic axonal capacity to synthesize proteins, we stress the glial derivation of axonal and presynaptic RNAs and the related proposal that these neuronal domains are endowed with largely independent gene expression systems (as reported by Giuditta et al. in Physiol Rev 88:515-555, 2008).

Synaptosomal protein synthesis is selectively modulated by learning.

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.

Permanent brain ischemia induces marked increments in hsp72 expression and local protein synthesis in synapses of the ischemic hemisphere.

Transient focal ischemia induced in rat brain by occlusion of the middle cerebral artery (MCAo) elicits a generalized induction of the 72 kDa heat-shock protein (hsp72) heralding functional recovery. As this effect implies activation of protein synthesis, and local systems of protein synthesis are present in brain synapses, and may be analyzed in preparations of brain synaptosomes, we evaluated hsp72 expression and protein synthesis in synaptosomal fractions of spontaneously hypertensive rats (SHRs) subjected to permanent MCAo. SHRs were randomly divided in ischemics and sham controls, anaesthesia controls and passive controls. Focal ischemia was induced under chloral hydrate anaesthesia by unilateral permanent MCAo. Protein synthesis was determined by [35S]methionine incorporation into synaptosomal proteins from ischemic and contralateral cortex/striatum, and from cerebellum. Hsp72 expression was measured in the same fractions by immunoblotting. Our data demonstrate that under these conditions synaptic hsp72 markedly increases in the ischemic hemisphere 1 and 2 days after MCAo, progressively declining in the following 2 days, while no significant change occurs in control rats. In addition, in the ischemic hemisphere the rate of synaptic protein synthesis increases more than two-fold between 1 and 4 days after MCAo, without showing signs of an impending decline. The present data provide the first demonstration that synaptic protein synthesis is massively involved in brain plastic events elicited by permanent focal ischemia.

Axonal and presynaptic RNAs are locally transcribed in glial cells.

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.

Brain metabolic DNA in memory processing and genome turnover.

Sophisticated methods are currently used to investigate the properties of brain DNA and clarify its role under physiological conditions and in neurological and psychiatric disorders. Attention is now called on a DNA fraction present in the adult rat brain that is characterized by an elevated turnover and is not involved in cell division or DNA repair. The fraction, known as brain metabolic DNA (BMD), is modulated by strain, stress, circadian oscillations, exposure to enriched or impoverished environment, and notably by several training protocols and post-trial sleep. BMD is frequently localized in glial cells but is also present in neurons, often in the perinucleolar region. Its distribution in repetitive and non-repetitive DNA fractions shows that BMD differs from native DNA and that in learning rats its profile differs from that of control rats. More detailed knowledge of the molecular, cellular, and time-dependent BMD features will be necessary to define its role in memory acquisition and processing and in the pathogenesis of neurologic disorders.

Axonal and presynaptic protein synthesis: new insights into the biology of the neuron.

The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal -- yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins. To dispel this lingering neglect, we now present the wealth of recent observations bearing on this central idea, and consider their impact on our understanding of the biology of the neuron. We demonstrate that extrasomatic translation sites, which are now well recognized in dendrites, are also present in axonal and presynaptic compartments.

Brain and behavioural evidence for rest-activity cycles in Octopus vulgaris.

Octopus vulgaris maintained under a 12/12h light/dark cycle exhibit a pronounced nocturnal activity pattern. Animals deprived of rest during the light period show a marked 'rebound' in activity in the following 24h. 'Active' octopuses attack faster than 'quiet' animals and brain activity recorded electrically intensifies during 'quiet' behaviour. Thus, in Octopus as in vertebrates, brain areas involved in memory or 'higher' processes exhibit 'off-line' activity during rest periods.

Is it only neurogenesis?

For a long time, the occurrence of neurogenesis in the adult mammalian brain was deemed non-existent or, at best, restricted to phylogenetically old brain regions. The pendulum of current opinion has now swung in the opposite direction with growing awareness that incorporation of labeled precursors into neuronal DNA occurs widely in the brain, and undergoes significant modulation with learning, different kinds of experiential inputs, and a number of physiological manipulations. A thorough review of the literature indicates that unscheduled DNA synthesis may significantly contribute to available evidence. Notably, data interpreted in terms of nerve cell turnover are more likely to reflect turnover of neuronal DNA, as suggested by earlier investigations.

Waking EEG power spectra in the rat: correlations with training performance.

Adult rats chronically implanted with supradural electrodes were telemetrically EEG recorded during a baseline session, a training session for a two-way active avoidance task, and a retention session. Rats were assigned to a fast learning (FL), slow learning (SL) and non learning (NL) group if they achieved criterion during the training session, the retention session, or in neither session. High-resolution EEG analyses indicated that intergroup differences were present in the low frequency range of waking baseline power spectra. Moreover, baseline delta emissions directly correlated with freezings, and inversely correlated with avoidances, while emissions at 7-10 Hz directly correlated with avoidances and inversely correlated with freezings. Interestingly, during the first training period, waking delta emission selectively increased in FL rats in concomitance with a marked performance improvement; instead, SL and NL rats displayed increments at 7-9 Hz. In addition, freezings scored during the first two training periods directly correlated with post-training waking emission at 2 Hz, and inversely correlated with emission at 7-10 Hz. Conversely, escapes and avoidances directly correlated with waking emission at 7-10 Hz. The data indicate that (i) waking baseline power spectra differ among behavioral groups, and correlate with behavioral performance the following day; (ii) selective modifications of waking power spectra occur in each behavioral group during training; and (iii) behavioral responses during training correlate with post-training waking power spectra. Notably, the delta increment selectively occurring in training FL rats is assumed to reflect online memory processing leading to better performance. The latter observation supports the primary involvement of delta waves in learning.