Antonio Giuditta

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.

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.

Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm

Abstract: Axoplasmic RNA from the giant axon of the squid (Loligo pealii)comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/μg of total RNA) suggests that the poly(A)+ RNA population makes up ∼0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments < 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower‐reacting component (moderately abundant and rare sequences). The latter component accounts for ∼56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of ∼2.7 kilobases and 3.1 × 102 kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents ∼0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

The sequential hypothesis on sleep function. I. Evidence that the structure of sleep depends on the nature of the previous waking experience.

The sequential hypothesis on sleep function assumes that the information gathered by brain during the waking period is processed during sleep in two main steps occurring during synchronized sleep (SS) and, eventually, during paradoxical sleep (PS). To verify the main consequences of the hypothesis, i.e., (1) that SS is involved in brain information processing; and (2) that the structure of sleep is dependent on the nature of the previous waking experience, an experiment was designed involving rats exposed to a training session (two-way active avoidance) but failing to learn (NL), and rats left in their home cages in the same training room (C). The structure of sleep, determined by EEG techniques in the postacquisition period (3 hr), was different in NL rats in comparison to C rats, chiefly because SS episodes were markedly longer in the former group. A more detailed analysis indicated that, in NL rats, SS episodes not followed by PS increased their duration first, while those followed by PS became longer in the second half of the sleep period. Comparable results were obtained in the comparison of NL and C subgroups deprived of PS at the end of the acquisition period by chlomipramine treatment. The data support the sequential hypothesis and provide evidence for a primary role of SS in brain information processing.

Local Gene Expression in Axons and Nerve Endings: The Glia-Neuron Unit

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.

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 [35S]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.

β-Actin and β-Tubulin are components of a heterogeneous mRNA population present in the squid giant axon

Abstract Previously, we have reported that the squid giant axon contains a heterogeneous population of polyadenylated mRNAs, as well as biologically active polyribosomes. To define the composition of this unique mRNA population, cDNA libraries were constructed to RNA obtained from the axoplasm of the squid giant axon and the parental cell bodies located in the giant fiber lobe. Here, we report that the giant axon contains mRNAs encoding β-actin and β-tubulin. The axonal location of these mRNA species was confirmed by in situ hybridization histochemistry, and their presence in the axoplasmic polyribosome fraction was demonstrated by polymerase chain reaction methodology. Taken together, these findings establish the identity of two relatively abundant members of the axonal mRNA population and suggest that key elements of the cytoskeleton are synthesized de novo in the squid giant axon.

Neurofilament Proteins Are Synthesized in Nerve Endings from Squid Brain

Abstract: It is generally believed that the proteins of the nerve endings are synthesized on perikaryal polysomes and are eventually delivered to the presynaptic domain by axoplasmic flow. At variance with this view, we have reported previously that a synaptosomal fraction from squid brain actively synthesizes proteins whose electrophoretic profile differs substantially from that of the proteins made in nerve cell bodies, axons, or glial cells, i.e., by the possible contaminants of the synaptosomal fraction. Using western analyses and immunoabsorption methods, we report now that (a) the translation products of the squid synaptosomal fraction include neurofilament (NF) proteins and (b) the electrophoretic pattern of the synaptosomal newly synthesized NF proteins is drastically different from that of the IMF proteins synthesized by nerve cell bodies. The latter results exclude the possibility that NF proteins synthesized by the synaptosomal fraction originate in fragments of nerve cell bodies possibly contaminating the synaptosomal fraction. They rather indicate that in squid brain, nerve terminals synthesize NF proteins.

The Structure of Sleep is Related to the Learning Ability of Rats

Using electroencephalographic methods, rats learning or not learning a two‐way active avoidance task were found to differ significantly in the structure of sleep determined the day before training. The main differences concerned (i) synchronized sleep episodes followed by wakefulness, which were longer and fewer in learning rats; (ii) paradoxical sleep episodes, which were longer in learning rats. Significant correlations were present between the number and/or the average duration of synchronized sleep episodes followed by wakefulness or by paradoxical sleep and the number of avoidances or escapes scored in the training session. Power spectral analysis indicated that the relative output in the 6 – 7‐Hz region was higher in learning rats, notably during short episodes of synchronized sleep followed by paradoxical sleep. As two‐way active avoidance training induces comparable modifications in postacquisition sleep (Ambrosini et al., Physiol. Behav., 51, 217 – 226, 1992), the features of preacquisition sleep which prevail in learning rats might directly determine their capacity to learn. Alternatively, they might reflect the existence of a genetic determinant independently conditioning the ability to learn.

Kinesin mRNA Is Present in the Squid Giant Axon

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β‐actin and β‐tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.