Carla Perrone Capano

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.

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.

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.

Local gene expression in nerve endings

At the Nobel lecture for physiology in 1906, Ramón y Cajal famously stated that “the nerve elements possess reciprocal relationships in contiguity but not in continuity,” summing up the neuron doctrine. Sixty years later, by the time the central dogma of molecular biology formulated the axis of genetic information flow from DNA to mRNA, and then to protein, it became obvious that neurons with extensive ramifications and long axons inevitably incur an innate problem: how can the effect of gene expression be extended from the nucleus to the remote and specific sites of the cell periphery? The most straightforward solution would be to deliver soma‐produced proteins to the target sites. The influential discovery of axoplasmic flow has supported this scheme of protein supply. Alternatively, mRNAs can be dispatched instead of protein, and translated locally at the strategic target sites. Over the past decades, such a local system of protein synthesis has been demonstrated in dendrites, axons, and presynaptic terminals. Moreover, the local protein synthesis in neurons might even involve intercellular trafficking of molecules. The innovative concept of glia‐neuron unit suggests that the local protein synthesis in the axonal and presynaptic domain of mature neurons is sustained by a local supply of RNAs synthesized in the surrounding glial cells and transferred to these domains. Here, we have reviewed some of the evidence indicating the presence of a local system of protein synthesis in axon terminals, and have examined its regulation in various model systems.

Messenger RNAs in synaptosomal fractions from rat brain.

Abstract 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.

The Neurochemical Study of Sleep

Knowledge of the complex relationship existing between sleep and brain biochemistry has been accumulating at a relatively slow pace. Progress in this field was thoroughly reviewed approximately 6 years ago,1,2 and there is little evidence that the rate of this progress has accelerated at all since then. Yet, such a goal should be considered an essential endeavor of the neurosciences. Although neurochemical studies cannot be taken as the only methodology leading to the goal, it is becoming increasingly evident that the final sentence of the chapter describing the role of sleep will not be written until we will know the type of operations a sleeping brain is performing at a molecular level. In turn, it is just as obvious that to design rewarding experiments at a molecular level, a general understanding of the biology of sleep is required, and questions should be asked within the framework of available hypotheses on the role of sleep. One should also remember that brain is not the only target organ for the functions of sleep. Other bodily activities are influenced by sleep, as is mental performance.

Local Protein Synthesis in the Squid Giant Axon and Presynaptic Terminals

The existence of a local system of axonal protein synthesis was proposed approximately 35 years ago to explain the similar rates of reappearance of acetylcholinesterase activity in proximal and distal regions of the cat hypoglossal nerve following irreversible inactivation of the enzyme (Koenig and Koelle, 1960). Since then this question has been approached with more direct techniques, by measuring the incorporation of radiolabelled aminoacids into the proteins of the axonal compartment following its separation from the closely apposed glial cells by microdissection or by autoradiographic methods (Koenig, 1984; Giuditta et al., 1990). The separation of axonal and glial compartments is more readily accomplished in large axons, such as the squid giant axon (Giuditta et al., 1968; 1983; 1991) and the Mauthner axon of the goldfish (Koenig, 1979; 1991; Koenig and Martin, 1996).

Gene Expression in Axons and Nerve Endings

According to an opinion still generally held (1), the axonal domain of a neuron (that is the axon and the nerve terminals) is lacking the capacity to synthesize proteins, with the obvious exception of the few proteins made in mitochondria. As a consequence, axonal and presynaptic proteins are assumed to be exclusively synthesized in the neuronal cell body, and to reach their final destinations by means of the axoplasmic transport.

Turnover of Cerebral DNA in Learning and Sleep

The possible involvement of brain DNA in learning received consideration some time ago, when nucleic acids appeared to be suitable candidates for the role of information storage molecules (Hyden, 1959; Gaito, 1961). DNA however was quickly put aside in view of its role as depository of genetic information and because of its presumed metabolic stability (Briggs and Kitto, 1962). On these counts RNA remained as a better candidate, at least until it was shown that brain had the same major RNA components of other organs (Vesco and Giuditta, 1967), thus leaving little room for brain specific RNA memory molecules. More convincing hypotheses were then advanced and RNA acquired a new role in learning as intermediate in the process of protein synthesis (Briggs and Kitto, 1962). It is ironical (and relevant) to note that both conclusions were based on incorrect assumptions. Indeed, a large population of brain-specific RNA transcripts came to be discovered in the early seventies following the application of hybridization techniques (Hahn and Laird, 1971; Brown and Church, 1971; Grouse et al., 1972). Their role in brain activity remains still to be clarified (Chaudari and Hahn, 1983; Kaplan, 1983). As to DNA, our views on its functional capacities have now become substantially more flexible in order to accomodate newly discovered features such as amplification, transposition, magnification and genomic rearrangement (Lewin, 1983).