J. R. Sotelo (PhD). Head of the Laboratory
J. C. Benech (PhD). Associate Professor
A. Kun (PhD). Research Assistant, Associated Unit to the Department of Biochemistry of de School of Sciences.
A. Calliari (PhD student). Research fellow
C. Escande (Student). Project fellowship (supporting agency, CSIC)
J. R. Sotelo-Silveira (PhD). Research collaborator (honorary), Research Assistant (Department of Molecular and Cell Neurobiology, IIBCE).
Students: L. Otero, M. Cárdenas, Soledad Marton, M. Victoria Elizondo, Agostina Puppo, Nancy Ghan, Paula Arbildi (School of Sciences). F. Gutiérrez (School of Veterinary)
Collaboration with other laboratories
Dr. J. M. Verdes (Msci), Biophysics Laboratory, School of Veterinary.
Dr. C. Negreira (PhD), Department of Physics, School of Sciences.

3) Research (brief description of the research subjects)

Three fundamental subjects were developed in our laboratory in the above mentioned time period: a) local protein synthesis in the axonal territory; b) calcium homeostasis including regulation by calcium of gene expression; c) local protein synthesis in the synaptic territory and its regulation by calcium: its relevance in synaptic plasticity. The literature included in this report represents examples of part of the work developed aiming to achieve the three above mentioned goals.
a) LOCAL PROTEIN SYNTHESIS IN THE AXONAL TERRITORY.
The matter of whether the axonal territory is able to synthesize its own proteins, has been considered a controversial issue since long time ago. The origin of this controversy can be found in the few opportunities that conventional electron microscopy showed ribosomes or polysomes in axons. In this regard, our Lab has been involved for a long period of time in this controversy. As it can be read in the List of Publication (LP), five of the eight publication of this period (3, 4, 6, 7, 8) dealt with this subject. Since the publication of one of the formers autoradiographic demonstrations of RNA and protein synthesis in the axonal territory of mammals (Benech C, Sotelo JR, et al., Exp. Neurol. 1982) our lab dedicated its time and the financial support got, to improve the tools able to answer so fundamental question. In this regard, the three neurofilament subunits (neuron specific proteins) were amongst those proteins we demonstrated that are newly synthesized in response to nerve injury (4, LP). The mRNA coding for these three proteins are present in intact nerves, but its expression is enhanced in response to injury in the proximal as well as the distal nerves stumps. We developed a polyclonal antibody against ribosomes that revealed ribosomes linked to the cytoskeleton of the squid giant axon and giant synapse (3, LP). Using this or other commercial antibodies, we find a preferential cortical subaxolemal localization of ribosomes (less than 1 ?m width zone) in vertebrate axons (4, LP and 1, in redaction, LP). Both circumstances could explain the characteristic elusiveness of ribosomes in axons. These contributions were inserted in a steady flow of information generated by few laboratories in the world that accumulated a bulk of evidences showing that the axonal territory is able to synthesize at least some of their own proteins. To get an exhaustive insight of the subject is important to read some review articles such as the one written by Alvarez, Koenig, Giuditta, (Prog. Neurobiol., 62:1-62, 2000).
E. Koenig in the early sixties, developed a technique for desheathing axons manually, using clockmaker tweezers. He prepared what he called myelin free axon whole-mounts. In 1996 Koenig and Martin, staining myelin free axon with Yoyo-1, discovered a subaxolemal structure in the axonal cortex tightly related to actin cytoskeleton that are full of RNA. Phosphorous Electron Spectroscopic Imaging (ESI) showed that ribosomes and polysomes are enriched in these regions (6, LP). Regarding this, Koenig named them as Periaxoplasmic Ribosomal Plaques (PARPs). In collaboration with Koenig, Sotelo-Silveira (from our lab), found that beta actin encoding mRNA is also enriched in PARPs (in situ hybridization, RT-PCR, manuscript in redaction). Furthermore, also rRNA was detected in PARPs. It was detected using the monoclonal antibody Y10B, obtained from an autoimmune disease. Similar results were obtained in ventral root axons of rats and rabbits.
Meanwhile in our lab, an actin motor protein, Myosin V, felt on our focus of interest.
Myosin Va is locally synthesized in nerves and its rate of synthesis is enhanced after nerve injury, like neurofilaments (immuno-absortion, 7, LP). The expression of Myosin Va mRNA is up regulated after nerve injury (7, LP). The cortical localization of Myosin V in axons prompted both labs, to investigate its possible relationship with PARP domains. PARP domains of axonal whole-mounts of rat and rabbit ventral roots and Mauthner axons are enriched in Myosin Va, as well as, Kinesin (KIF 3A motor protein, 8, LP). Finally, mRNA coding for Myosin Va also was found enriched in PARP domains. Other protein, HuD (an RNA binding protein) was found enriched in PARP domains (to be published elsewhere). The presence of ribosomes, at least two mRNAs, an RNA binding protein as HuD and two motor proteins as Myosin Va and Kinesin in PARP domains, suggested us, that they could be local centers of protein synthesis. Probably, they are involved in axonal maintenance, regeneration and/or plasticity. Besides the latter, the localization of all this molecules, especially mRNAs, in PARP domains, so far of their nuclei of origin, also suggests the dynamic nature of these structures. They may do interchange with the surrounding axoplasm, but also they would receive ribosomes and mRNAs from the neuronal soma. We think that newly synthesized proteins will be distributed from these local centers (PARPs). Furthermore, all the above mentioned information about PARPs and Local Protein Synthesis should be important to determine neuronal polarity. Neurons are cells that have a high level of structural polarity. To get this polarity they need a complex system of transport of subcellular components toward dendritic and postsynaptic domains, or toward axonal and presynaptic domains. Part of the proteins of dendrites or axons is synthesized in the perikarion, but other is locally synthesized. The latter depends on RNA transport and is one of the matters of growing interest now. The molecular composition of these proteins, define different domains. So there are dendritic proteins or axonal proteins.
The main goal we have in mind, regarding our future research, is to unravel the composition of RiboNucleoParticles (RNP) transported into axons and its relationship to PARPs. RNA targeting and the composition of RNP as well as their relation to motor proteins will be important clues to understand the regulation of mRNA expression and its participation in determining neuron polarity, as well as, regeneration and plasticity.

b) CALCIUM HOMEOSTASIS INCLUDING REGULATION BY CALCIUM OF GENE EXPRESSION; c) LOCAL PROTEIN SYNTHESIS IN THE SYNAPTIC TERRITORY AND ITS REGULATION BY CALCIUM: its relevance in synaptic plasticity.
Our Lab is interested since long time ago in the participation of Local Protein Synthesis of the synaptic territory, in a process like synaptic plasticity. In the last 10 years Local Protein Synthesis has been demonstrated to be absolutely necessary for synaptic plasticity at least in Aplysia (Martin and Kandel, et al., Cell), and axonal guidance during axonal outgrowth in the Central Nervous System of vertebrates (Brittis et al., 2002). Regarding this, Benech, J. C., who has a long experience in calcium homeostasis (1, LP), collaborated with A. Giuditta, to determine if Calcium, -a second messenger responsible of triggering neurotransmitter release in presynaptic nerve terminals-, could modulate local protein synthesis in the synaptic territory (2, LP). They demonstrated that protein synthesis of synaptosomes prepaired from squid optic lobe, is dependent of cytosolic free [Ca2+] and it has its optimal rate when the free calcium concentration is near to 100 nM (resting calcium). When cytosolic free calcium concentration is lowered below resting calcium or increased above resting calcium, synaptosomal protein synthesis is inhibited. These results prompted us to measure cytosolic free [Ca2+] in optic lobe synaptosomes in collaboration with E. Brown (UK). Free [Ca2+] was measured using Fura-2 AM and Calcium Green AM in single optic lobe synaptosomes (5, LP), in metabolic conditions resembling those used to measure synaptosomal protein synthesis. Measurements confirmed that synaptosomes were alive and responsive to calcium homeostasis changes induced by Ca2+ channels opening. Synaptosomes, also have calcium internal stores controlled by SERCA pumps. It also was confirmed that changes in calcium concentration could be the origin of the above mentioned protein synthesis modulation.
Finally, Benech et al., have developed a new interesting paradigm, consisting in an isolated nuclei preparation to study the relationship between RNA synthesis and the Ca2+ filled state of the nuclear envelope. Regarding this paradigm, they found that isolated nuclei are able to synthesize RNA, and this synthesis is dependent on the free [Ca2+] of the nucleoplasm, but also by the Ca2+ filled state of the nuclear envelope (one of the so-called internal calcium stores). Moreover, the Ca2+ filled state of the Nuclear Envelope Store (NES) has been demonstrated to be involved in the opening of the Nuclear Pore Complex (Perez-Terzic, et al., Science, 1996), which is well known to control the nuclear-cytoplasmic molecular traffic. One of the aims of this research is to understand which genes are turn on or turn off throughout changes of nucleoplasmic [Ca2+], induced by local stimulus or stimulus coming from cell environment (2, in redaction, LP). In this regard, they are also studying the effect of changes of nuclear calcium homeostasis on the expression of genes containing early response sequences such as cAMP Responsive Elements (CRE) that has been found to be involved in nuclear calcium signaling.

4) List of Publications

1-R. García-Tejeiro, J. R. A. Sotelo-Silveira, J. R. Sotelo, J. C. Benech. Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the calcium pump. Molecular and Cellular Biochemistry. 199(1-2):7-14.

2- Benech, J.C, Crispino, M, Kaplan, B.B. and Guiditta, A. Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions. Journal of Neuroscience Research (55):776-781.

3- J. R. Sotelo, A. Kun, J. C. Benech, J. Morillas, A. Giuidtta, C. R. Benech. Ribosomes and Polyribosomes are Present in the Squid Giant Axon: an Immunocytochemical study. Neuroscience, 90(2):705-715.

4- J. R. A. Sotelo-Silveira, A. Calliari, A. Kun, J. C. Benech and J. R. Sotelo. Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve. Journal of Neuroscience Research, 62:65-75.

5- J. C. Benech, P. Lima, C. Rogers, J. R. Sotelo, E. Brown. Ca2+ dynamics in synaptosomes isolated from the squid optic lobe. Journal of Neuroscience Research, 62:840-846.

6- -Koenig E, Martin R, Titmus M, Sotelo-Silveira J. R. Cryptic peripheral ribosomal domains distributed intermittently along mammalian myelinated axons. Journal of Neuroscience 20(22):8390-400.

7- A. Calliari, M. C. Costa, J. Nogueira, L.C. Cameron, A. Kun, J. R. Sotelo Silveira, J. C. Benech, J. R. Sotelo. Myosin Va is locally synthesized following nerve injury. Cell Motility and the Cytoskeleton, 51:169-176.

8- J. R. Sotelo-Silveira, A. Calliari, M. Cárdenas, E. Koenig and J. R. Sotelo. Myosin Va and Kinesin motor proteins are concentrated in ribosomal domains (periaxoplasmic ribosomal plaques) of myelinated axons. Journal of Neurobiology.

In redaction

1- A. Kun, L. Otero, J. R. Sotelo. Ribosomes and polysomes detected in axons of peripheral nerves of vertebrates: an immunocytochemical study. In redaction.

2- Benech, J.C.; Escande, C. and Sotelo, J.R.. Relationship between RNA synthesis and the Ca2+ filled state of the nuclear envelope. In redaction.

5) Teaching and training activities

The lab participates in:

-pre-graduate courses of the School of Sciences and the School of Veterinary.
-post-graduate courses of the Program of Basics Sciences Development (PEDECIBA)
-outreach activities towards Primary and Secondary Schools. Divulgation of research.
-PhD: A. Kun (PEDECIBA),.
-PhD: J. R. Sotelo Silveira (PEDECIBA).
-Master of José Manuel Verdes (PEDECIBA).
-Dr. J. R. Sotelo and Dr. J. C. Benech are Professors of the Posgraduate Courses of the School of Veterinary.
-Participation of Dr. J. R. Sotelo as Professor of the Doctorate of Neurosciences of Sevilla, University Pablo de Olavide of Sevilla.

6) Services and outreach activities

-Eight science divulgation articles were published by members of the Lab in local diffusion Magazines.
-Edition of a Science Section, entitled: “Researchers of the Clemente Estable” on the Uruguayan Magazine “Posdata”. The aim of this section was to bring the readers a broad vision of the state of the art on our own country. 115 articles were published. Editors: J. R. Sotelo and F. Costa (both from IIBCE)
-J. R.Sotelo is reviewer of Neuroscience
-Eleven invited lectures were given in the period.
-J. C. Benech, A. Kun and J. R. Sotelo, are professors of the Latinoamerican School of Neurosciences.
-J. C. Benech, A. Kun and J. R. Sotelo, were organizers of IV Latinoamerican School of Neurosciences.