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MECHANISMS OF NEURODEVELOPMENT
IN HEALTH AND DISEASE
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Phillip Nelson, MD, PhD, Head, Section on Neurobiologya Min Jia, MD, Senior Research Assistant Thea Kuddo,
MD, Guest Researcher Mara Angel Lanuza, PhD, Guest Researcher Min-Xu Li, MD, Guest Researcher Eun Young Song, PhD, Guest Researcher Rahel Gizaw,
Postbaccalaureate Fellow Cassandra VanDunk, BS, Postbaccalaureate Fellow Veronica Dunlap, Senior Technician |
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We have continued our studies
on the influence of activity on the development, stabilization, and
elimination of synapses in in vitro models
and in the intact organism. Previous work documented the rather complex
involvement of protein kinases A and C in the process of activity-dependent
synapse elimination at the rodent neuromuscular junction. Activity-dependent
synapse loss and stabilization at the neuromuscular junction Nelson P, Jia, Li, Lanuza, Gizaw Both kinases A and C were
involved in our in vitro system in
a mutually antagonistic manner both pre-and post-synaptically. While either a
pharmacological or genetic ablation of PKC produced an essentially permanent
block of activity-dependent synapse loss in
vitro, the process of loss in vivo
was delayed by over three days, but not permanently blocked. Evidently, in vivo the PKC plays a role in the
elimination process, but redundant mechanisms can mediate synapse loss during
development, even in the absence of PKC activity. During the process of synapse
loss, there is loss of the post-synaptic acetylcholine receptor (AChR), which
can be brought about by PKC action. Indeed, evidence suggests that receptor
loss may be an initiating event in the synapse loss process. The AChR is a
phosphoprotein that is a target of both PKA and PKC. Phosphorylation of the
epsilon PKA–specific site results in receptor stabilization while
phosphorylation of the delta subunit on PKC-specific sites results in
receptor destabilization. We developed some
antibodies against the phosphorylated and nonphosphorylated
forms of the AChR and used them to study whether a shift in the state of
phosphorylation of the AChR occurs under conditions in which we know there is
a change in receptor stability. We prepared the antibodies by immunizing
rabbits with the phosphorylated forms of peptide sequences containing known
phosphorylation sites for the two kinases. The antisera were run over a
column containing the nonphosphorylated peptides,
with the flow throughput over a column of the phosphorylated peptide. Bound
material was then eluted and treated as phospho-specific antibody. We found
that staining with the phospho-specific antibody against the epsilon subunit
was enhanced by treatment of nerve-muscle co-cultures with cAMP (which increases PKA activity). Staining with
phospho-specific antibodies against the delta subunit was more intense in
PMA-treated co-cultures (PMA increases PKC levels) than in electrically
silenced, TTX-treated cultures. Less expected was an increase in
phospho-epsilon staining under PMA treatment, perhaps resulting from a
decrease in epsilon nonphospho staining because of
its destabilization by the PMA-induced increase of
phosphorylation of the delta subunit. Li M-X, Jia
M, Yang L-X, Jiang H, Lanuza
MA, Gonzalez CM, Nelson PG. The role of protein kinase C (PKC) in
activity-dependent synapse elimination: evidence from the PKC theta knock-out
mouse in vivo and in vitro. J Neurosci 2004;24:3762-3769. Nelson PG, Jia M, Li M-X, Gizaw R, Lanuza M, Tomas J. In: Shimazu K, Takeda K, Yu Z-X, Jiang H, Liu X-W, Nelson PG, Guroff G. Multiple acute effects on the membrane
potential of PC12 cells produced by Nerve Growth Factor (NGF). J Cell Physiol, in press. Autocrine function for
GDNF at the neuromuscular junction Yang, Nelson P The trophin Glia Derived
Neurotrophic Factor (GDNF) is known to be produced by muscle and to have
powerful effects on spinal motor neurons and other nerve cells throughout the
brain. We have examined the possibility that GDNF also has an effect on the
muscle cell itself, in particular on the metabolism and membrane localization
of the acetylcholine receptor (AChR). We found that GDNF does indeed increase
the insertion rate of AChR into the surface membrane of muscle cells, with a
lesser effect on receptor loss from the membrane and without any appreciable
effect on receptor synthesis. Several inhibitor studies suggest that the GDNF
effect is mediated by the alpha 1 GDNF receptor and involves the Ret
receptor, MAP kinase, cAMP/CREB, and Src kinase activity. Thus, the trophin may act in a
synergistic pre- and postsynaptic manner to modify synapse efficacy. Yang L-X, Nelson PG.
Neurotrophic factor GDNF regulates the distribution of acetylcholine
receptors in primary muscle cells. Neuroscience
2004;128:497-509. Molecular basis for
neurodevelopmental disorders Nelson P, Song, Kuddo, VanDunk; in
collaboration with Grether, Nelson K We have extended our study of
archived samples from control children and those with subsequently diagnosed
developmental disorders to include children with Down syndrome (DS). We found
that in newborns with DS the level of the cytokine IL-8 is significantly
elevated (higher by about 75 percent). In addition, the archived blood spots
revealed distinct developmental profiles for the analytes that we have
measured. The level of BDNF, for instance, is low in very premature infants,
higher in normally mature neonates, and highest in adult material. By
contrast, the levels of IL-8 are highest in the very premature, very low in
adults, and intermediate in the normal gestation newborns. We have developed
a novel competitive fluorescent microsphere immunoassay for Vasoactive
Intestinal Peptide using the Luminex flow cytometry
platform. aRetired in June, 2004; now Scientist Emeritus. COLLABORATORS Judy Grether, PhD,
California Department of Health Services, Karin B. Nelson, MD, Neuroepidemiology Branch, NINDS, For further
information, contact pgnelson@codon.nih.gov |
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