Protein synthesis in synaptosomes: a proteomics analysis.

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Messenger RNAs in synaptosomal fractions from rat brain.

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.

Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal.

One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear-encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl-CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447-453, 2001. Published 2001 Wiley-Liss, Inc.

Trains of sleep sequences are indices of learning capacity in rats.

In previous work dealing with the identification of four sleep sequences (SS-->W, SS-->PS, SS-->TS-->W and SS-->TS-->PS) in the baseline session of adult male Wistar rats [Mandile P, Vescia S, Montagnese P, Romano F, Giuditta A. Characterization of transition sleep episodes in baseline EEG recordings of adults rats, Physiol Behav 1996;60:1435-1439], we have shown that those containing an intervening episode of transition sleep (TS) strongly correlate with the number of avoidances scored the following day [Vescia S, Mandile P, Montagnese P, Romano F, Cataldo G, Cotugno M, Giuditta A. Baseline transition sleep and associated sleep episodes are related to the learning ability of rats, Physiol Behav 1996;60:1513-152]. More recently, clusters of sleep sequences (trains) separated by waking intervals longer than 60 s have been identified in the baseline session of the same rats [Piscopo S, Mandile P, Montagnese P, Cotugno M, Giuditta A, Vescia S. Identification of trains of sleep sequences in adult rats, Behav Brain Res, this volume], and distinguished in homogeneous or mixed trains according to the presence of a single sleep sequence or more than one sequence. Mixed trains have been further separated into trains containing the SS-->TS-->W sequence (+TSW trains) and trains lacking it (-TSW trains). Analysis of the distribution of variables of baseline trains (and of their sleep sequences and components) among fast learning (FL), slow learning (SL), or non-learning (NL) rats, indicates that variables of +TSW trains prevail in FL rats, while variables of -TSW trains prevail in NL rats. In addition, variables of +TSW trains correlate with the number of avoidances of the training session, while variables of -TSW trains do not significantly correlate, or show inverse correlations. Interestingly, sleep sequences such as SS-->W or SS-->TS-->W show direct or inverse correlations with avoidances depending on whether they are included in +TSW trains or in -TSW trains. The data are interpreted to suggest that the outcome of brain operations performed during a sleep sequence may selectively condition the appearance of later sequences within a time interval shorter than a given threshold. An analogous mechanism may be responsible for the aggregation of sleep components in sleep sequences.

Identification of trains of sleep sequences in adult rats.

In previous studies based on high resolution EEG analyses of the 7 h baseline session of 18 adult male Wistar rats [6,14], we have identified four sleep sequences initiating with slow wave sleep (SS) and terminating with waking (W) or paradoxical sleep (PS). Two of these sequences contained an intervening episode of transition sleep (TS). Several variables of these sequences (SS-->W, SS-->TS-->W, SS-->TS-->PS, and SS-->PS) were selectively correlated with the capacity of rats to learn a two-way active avoidance task the following day, and were differently distributed in fast learning, slow learning and non learning rats [21]. The temporal organization of different sleep components in sequences suggested that a comparable temporal organization might concern the different sleep sequences, albeit on a longer time scale. We have now used waking periods longer than 60 s to separate clusters of baseline sleep sequences (trains) in the same rats. Trains containing the same sleep sequence (homogeneous trains) have been distinguished from trains containing different sleep sequences (mixed trains). In addition, mixed trains including the SS-->TS-->W sequence (+TSW trains) have been separated from mixed trains lacking that sequence (-TSW trains). Mixed trains of the +TSW type were longest and most numerous, while homogeneous trains were shortest and least abundant. Mixed trains of the -TSW type displayed intermediate values. Several variables of sleep sequences and sleep components differed within mixed trains and among mixed and homogeneous trains. The data indicate that baseline sleep sequences aggregate in relatively long strings in a non random fashion. The mechanism of this association is discussed.

Learning and sleep: the sequential hypothesis.

During the last 30 years, paradoxical sleep (PS) has been generally considered as the only type of sleep involved in memory processing, mainly for the consistent increase of PS episodes in laboratory animals learning a relatively complex task, and for the retention deficits induced by post-training PS deprivation. The vicissitudes of this idea, examined in detail by several laboratories, have been critically presented in a number of review articles However, according to a more comprehensive unitary proposal (the sequential hypothesis), memory processing during sleep does require the initial participation of slow-wave sleep (SS) in addition to the subsequent involvement of PS. The evidence supporting this hypothesis, largely derived from experiments concerning rats trained for a two-way active avoidance task, is reviewed here in some detail. Recent studies of human sleep are in full agreement with this view. In the rat, the main effect of learning on post-training SS consists in the selective increment in the average duration of SS episodes initiating different types of sleep sequences. Notably, following training for a two-way active avoidance task, the occurrence of this effect in sleep sequences including transition sleep (TS), such as SS-->TS-->W and SS-->TS-->PS, appears related to the processing of memories of the novel avoidance response. Conversely, the occurrence of the same effect in sleep sequences lacking TS may reflect the processing of memories of innate responses (escapes and freezings). Memories of innate and novel responses are assumed to engage in a dynamic competitive interaction to attain control of waking behaviour. Interestingly, in baseline sleep, variables of SS-->TS-->W and SS-->TS-->PS sequences, such as the average duration of SS, TS, and PS episodes, have proved to be good indices of the capacity to learn, as shown by their strong correlations with the number of avoidances scored by rats the following day. Comparable correlations have not been displayed by variables of baseline SS-->W and SS-->PS sequences.

Post-trial sleep sequences including transition sleep are involved in avoidance learning of adult rats.

High resolution computerized EEG analyses, and behavioral observations were used to identify slow wave sleep (SS), paradoxical sleep (PS) and transition sleep (TS) in adult male Wistar rats exposed to a session of two-way active avoidance training. Of the four sleep sequences that could be identified, two included TS (SS-->TS-->W and SS-->TS-->PS), while the other two did not (SS-->W and SS-->PS). Comparison of post-trial sleep variables between fast learning rats (FL, reaching criterion in the training session), slow learning rats (SL, reaching criterion in the retention session the following day), and non learning rats (NL, failing to reach criterion) indicated that the total amounts of SS, TS and PS of the SS-->TS-->PS sequence was markedly higher in FL rats than in SL rats. In addition, in comparison with the corresponding baseline period, the average duration and total amount of SS and TS episodes of the SS-->TS-->PS sequence increased in FL rats, while the number of SS-->TS-->W sequences decreased. On the other hand, the average duration of SS episodes increased in the SS-->TS-->W and SS-->W sequences of SL rats, and in the SS-->W and SS-->TS-->PS sequences of NL rats. Correlative analyses between number of avoidances and post-trial sleep variables demonstrated that avoidances were directly correlated with the duration of SS episodes of the SS-->TS-->PS sequence and with the duration of TS episodes of the SS-->TS-->W sequence, but inversely correlated with the number and amount of SS episodes of the SS-->W sequence and with the duration and amount of SS episodes of the SS-->PS sequence. On the whole, the data supported the view that TS-containing sleep sequences are involved in long-term storage of novel adaptive behavior, while sleep sequences lacking TS are involved in the maintenance of innate behavioral responses.

Protein synthesis in axons and terminals: significance for maintenance, plasticity and regulation of phenotype. With a critique of slow transport theory.

This article focuses on local protein synthesis as a basis for maintaining axoplasmic mass, and expression of plasticity in axons and terminals. Recent evidence of discrete ribosomal domains, subjacent to the axolemma, which are distributed at intermittent intervals along axons, are described. Studies of locally synthesized proteins, and proteins encoded by RNA transcripts in axons indicate that the latter comprise constituents of the so-called slow transport rate groups. A comprehensive review and analysis of published data on synaptosomes and identified presynaptic terminals warrants the conclusion that a cytoribosomal machinery is present, and that protein synthesis could play a role in long-term changes of modifiable synapses. The concept that all axonal proteins are supplied by slow transport after synthesis in the perikaryon is challenged because the underlying assumptions of the model are discordant with known metabolic principles. The flawed slow transport model is supplanted by a metabolic model that is supported by evidence of local synthesis and turnover of proteins in axons. A comparison of the relative strengths of the two models shows that, unlike the local synthesis model, the slow transport model fails as a credible theoretical construct to account for axons and terminals as we know them. Evidence for a dynamic anatomy of axons is presented. It is proposed that a distributed "sprouting program," which governs local plasticity of axons, is regulated by environmental cues, and ultimately depends on local synthesis. In this respect, nerve regeneration is treated as a special case of the sprouting program. The term merotrophism is proposed to denote a class of phenomena, in which regional phenotype changes are regulated locally without specific involvement of the neuronal nucleus.

Induction of cerebellar hsp72 in rats learning a two-way active avoidance task.

The involvement of brain heat shock proteins in learning was examined by Western analyses in rats trained for an active avoidance task, and in passive and active controls. Expression of the constitutive hsp73 was intense in brain, liver, and kidney of all rats. Conversely, expression of the inducible hsp72 occurred in the cerebellum of most trained rats, but not in passive or active controls. Significant correlations were present between avoidances and cerebellar scores determined 8 h after training. Induction of hsp72 may therefore be attributed to learning in the cerebellum, while in other brain regions, liver and kidney stress-related stimuli may play a prevalent role.

Protein synthesis in presynaptic endings from squid brain: modulation by calcium ions.

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366-374; Crispino et al. [1993b] J. Neurochem. 61:1144-1146; Crispino et al. [1997] J. Neurosci. 17:7694-7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein.

Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

Protein-synthesizing machinery in the axon compartment.

Contrary to the prevailing view that the axon lacks the capacity to synthesize proteins, a substantial body of evidence points to the existence of a metabolically active endogenous translational machinery. The machinery appears to be largely localized in the cortical zone of the axon, where, in vertebrate axons, it is distributed longitudinally as intermittent, discrete domains, called periaxoplasmic plaques. Studies, based on translation assays and probes of RNA transcripts in axon models such as the squid giant axon and selected vertebrate axons, provide evidence of locally synthesized proteins, most of which appear to be constituents of the slow axoplasmic transport rate groups. Metabolic and molecular biological findings are consistent with the view that the synthesis of proteins undergoing local turnover in the axonal compartment of macroneurons depends on the activity of an endogenous translational machinery. The documented presence of a metabolically active machinery in presynaptic terminals of squid photoreceptor neurons is also described. Finally, potential sources of axoplasmic RNAs comprising the machinery, which may include the ensheathing cell of the axon, as well as the cognate cell body, are also discussed.

Ribosomal RNAs synthesized by isolated squid nerves and ganglia differ from native ribosomal RNAs.

The large rRNA of the squid comprises two chains that may be dissociated by heating at 65 degrees C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonetheless, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was localized in nerve and glial cells, as shown by autoradiographic analysis, and was assembled into 80S ribosomes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for >10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32P-cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of homology with native rRNA in northern analysis. Taken together, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neuronal degeneration/regeneration.

Protein synthesizing units in presynaptic and postsynaptic domains of squid neurons.

Putative protein synthesizing domains, called plaques, are characterized in the squid giant synapse and axon and in terminals of squid photoreceptor neurons. Plaques are oval-shaped formations of about 1 microm in size, which (1) generate signals that have spectroscopic electron energy loss characteristics of ribosomes, (2) exhibit ribonuclease-sensitive binding of YOYO-1, a fluorescent RNA/DNA dye, and (3) in part hybridize with a poly(dT) oligonucleotide. In the giant synapse plaques are abundant in the postsynaptic area, but are absent in the presynaptic terminal. In the cortical layer of the optic lobes, plaques are localized in the large carrot-shaped presynaptic terminals of photoreceptor neurons, where they are surrounded by synaptic vesicles and mitochondria. Biochemical and autoradiographic data have documented that the protein synthetic activity of squid optic lobe synaptosomes is largely due to the presynaptic terminals of the photoreceptor neurons. The identification of ribosomes and poly(A+)-mRNA in the plaques indicates that these structures are sites of local protein synthesis in synaptic domains.

Post-trial sleep in old rats trained for a two-way active avoidance task.

Nine male Wistar rats aged 27 months were trained for a two-way active avoidance task and tested for retention the following day. At variance with young adult rats, most of which succeed in mastering the task, all old rats displayed a large majority of freezing responses throughout the training and the retention sessions, thereby confirming the condition of learning impairment of aged rats. Comparison of baseline and post-trial sleep indicated the presence of a transient, but marked, increment in the average duration and total amount of post-trial slow-wave sleep followed by waking, and of a decrease in total amount of quiet waking. On the other hand, variables of paradoxical sleep and of slow-wave sleep followed by paradoxical sleep or by transition sleep did not show significant variations. Because these sleep variables are known to undergo significant variations in learning in young adult rats, the present data confirm that the latter effects are related to memory-processing events rather than to nonspecific effects of training. An additional outcome of training consisted in a marked post-trial decrement in the number of spike-wave discharges, which are known to occur in old rats during periods of quiet waking.

Active polysomes are present in the large presynaptic endings of the synaptosomal fraction from squid brain.

Previous data have suggested that the large nerve terminals present in the synaptosomal fraction from squid optic lobe are capable of protein synthesis (Crispino et al., 1993a,b). We have further examined this issue by comparing the translation products of synaptosomal and microsomal polysomes. Both preparations programmed an active process of translation, which was completely abolished by their previous treatment with EDTA. After immunoabsorption of the newly synthesized neurofilament (NF) proteins, the labeling ratio of the 60 and 70 kDa NF proteins was found to differ, in agreement with comparable differences obtained with intact synaptosomes. These observations indicate that the set of mRNAs translated by synaptosomes differs from that translated by nerve cell bodies. Hence, because NF proteins are neuron-specific, they support the view that the active synaptosomal polysomes are mostly localized in the large nerve terminals that represent the most abundant neuronal component of the fraction. This hypothesis was confirmed (1) by electron spectroscopic data demonstrating the presence of ribosomes and polysomes within the large nerve endings of the synaptosomal fraction, as well as in the carrot-like nerve endings of the retinal photoreceptors that constitute the only large terminals in the optic lobe, and (2) by light and high resolution autoradiography of synaptosomal samples incubated with [3H]leucine, showing that most labeled proteins are associated with the large nerve endings. This response was abolished by cycloheximide. Taken together, the data provide the first unequivocal demonstration that presynaptic nerve terminals are capable of protein synthesis.

Molecular cloning and characterization of a novel mRNA present in the squid giant axon.

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.

Role of a transcription factor (CREB) in memory processes.

Memory storage includes a short-term phase (STM) which requires the phosphorylation of pre-existing proteins, and a long-term phase (LTM) which needs the novel synthesis of RNA and proteins. Cyclic AMP and a specific transcription factor (cAMP response element binding protein or CREB) play a central role in the formation of LTM in aplysia, drosophila and mice. Following its phosphorylation by protein kinase A, CREB binds to the enhancer element CRE which is located in the upstream region of cAMP-responsive genes, thus triggering transcription. Some of the newly-synthesized proteins are additional transcription factors that ultimately give rise to the activation of late response genes, whose products are responsible for the modification of synaptic efficacy leading to LTM. In aplysia, CREB activation has been interfered with by microinjection of CRE containing oligonucleotides into cultured neurons. Under these conditions LTM is blocked while STM remains unchanged. In drosophila, CREB function has been disrupted using a reverse genetic approach. Thus, LTM has been specifically blocked by the induced expression of a CREB repressor isoform, and enhanced by the induced expression of an activator isoform. In mouse, the role of CREB has been confirmed by behavioural analyses of a knock-out line with a targeted mutation in the CREB gene. In these mutants, learning and STM are normal, whereas LTM is disrupted. On the whole, the data suggest that encoding of long term memories involve highly conserved molecular mechanisms.

Characterization of transition sleep episodes in baseline EEG recordings of adult rats.

By scoring 5-s EEG epochs and calculating spectral power of consecutive EEG segments as short as 1-s, transition sleep (TS) episodes were identified in baseline recordings of adult rats. TS episodes were characterized by the abrupt appearance of theta and alpha waves within an ongoing period of slow-wave sleep (SS). They were followed by paradoxical sleep (PS) or, somewhat more frequently, by a period of wakefulness (W) that often led to an additional SS. Statistical values of the main variables of TS-->(W) and TS-->(PS) episodes are presented, together with comparable data concerning previous SS and following W or PS episodes. On the whole, TS episodes were more numerous than PS episodes, and less numerous than SS episodes. Their average duration was considerably shorter. As a consequence of the identification of TS and of brief W or PS epochs intervening within SS, the number of SS episodes was estimated to be considerably higher than previously assessed, and their average duration considerably shorter.