Laboratory
of Proteins and Nucleic Acids
Laboratory
of Proteins and Nucleic Acids
Collaboration
with other laboratories
Dr. J. M. Verdes (Msci), Biophysics Laboratory, School of
Veterinary.
Dr. C. Negreira (PhD), Department of Physics, School of
Sciences.
Three
fundamental subjects were developed in our laboratory in the above mentioned
time period (1999-2003):
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.
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, 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, 1997), 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.
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, 1999
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, 1999.
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, 1999.
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, 2000.
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, 2000.
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, 2000.
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, 2002.
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 press 2004.
In
redaction
1- A. Kun, L. Otero, J. R. Sotelo. 2004 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. 2004. Relationship between
RNA synthesis and the Ca2+ filled state of the nuclear envelope. In redaction.
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), 1999.
-PhD: J. R. Sotelo Silveira (PEDECIBA), 2003.
-Master of José Manuel Verdes (PEDECIBA), 2003.
-Dr. J. R. Sotelo and Dr. J. C. Benech are Professors of the Posgraduate Courses
of the School of Veterinary, (1999-2001, creation of the Program).
-Participation of Dr. J. R. Sotelo as Professor of the Doctorate of
Neurosciences of Sevilla, University Pablo de Olavide of Sevilla, 2003.
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, 1999.
Grants
1- Newly protein synthesis in rat brain synaptosomes: modulation by changes in
calcium homeostasis. J. C. Benech. Financial Agency (FA), CSIC 1998-1999. US$20,000.-
2- Molecular characterization of a new axonal domain Periaxoplasmic Ribosomal
Plaques (PARPs): its role in protein synthesis. CSIC (FA),
2000-2002. J. R. Sotelo- Silveira. US$11,000.-
4- Participation of Myosin V (BM-V) in Peripheral Nervous System regeneration
CIDEC (FA), 1999. US$2,000.-, Aldo Calliari.
5- Model of Science and Technology integration among Argentina, Brasil y
Uruguay. J. R. Sotelo. Organization of American States (OAS, FA), 2000. US$21,000.-
6- Model of Science and Technology integration among Argentina, Brasil y
Uruguay. II spatial and temporal enlargement of the original network. J.
R. Sotelo. (OAS, FA), 2001.
US$70,000.-
7- Cloning and characterization of translated genes in synaptic terminals. J.
R. Sotelo, J. C. Benech (Uruguay) y A. Carrión (Spain). Iberoamerican Cooperation Agency (FA), 2002. US$5,000.-
8- Cloning and characterization of translated genes in synaptic terminals. J.
R. Sotelo, J. C. Benech (Uruguay) y A. Carrión (Spain). Iberoamerican Cooperation Agency (FA), 2003. US$5,000.-
9- Calcium homeostasis in the cell nucleus. Role of the calcium gradient of the
nuclear envelope in isolated rat liver nuclei, its posible function in the
modulation of RNA synthesis. Third World Academy of Sciences (TWAS, FA). 18
months, 2002. U$6,000.- J. C. Benech.
10- Ca2+ Homeostasis in the
cell nucleus: Nuclear envelope Ca2+ATPasa. The role of Ca2+ filled state of the
Nuclear Envelope on RNA synthesis modulation. (CSIC, FA). J. C. Benech, 2002.
11- Southamerican nanobiotechnology and biomimetics systems network. CNPq
(FA), Brasil, 2004. J. C. Benech