Mechanisms of Neurodevelopment in health and disease

Child Health And Human Development

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Abstract

Summary of work:We have continued our work on the cell biological mechanisms involved in activity-dependent, Hebbian synapse elimination in the neuromuscular synaptic system. We have begun molecular studies of possible pathogenetic mechanisms involved in some human neurodevelopmental disorders. Synapse elimination in vitro and in vivo The Hebbian, activity-dependent modulation of synapse efficacy, in which activated pathways are strengthened and inactive ones weakened, depends on an integrated pattern of activation of protein kinases , such that appropriate phosphorylation reactions occur which differentially affect the stability of activated and non-activated synapses and their associated receptors. A step in the activation of the kinases, in particular Protein Kinase C (PKC), involves the action of the serine protease, thrombin, and its associated receptor, the protease activated receptor or PAR. Thrombin's action can be reproduced by a peptide ,the thrombin receptor activating peptide (TRAP) that can activate the PAR. Collaborative experiments with Dr. Lanuza and her group in Spain have shown that the thrombin receptor (ThR) is localized at the neuromuscular junction in vivo and that application of the TRAP increases the rate of synapse elimination that occurs naturally in their system. Thus, the molecular apparatus for thrombin's participation in the synapse modification process is present and functional at the neuromuscular junction. The natural elimination process can be divided into three stages: an early stage in which significant loss of polyneuronal innervation occurs with relatively little change in the distribution of post-synaptic receptors, an intermediate stage in which both axonal loss and receptor changes are prominent and a final stage in which the innervation has become primarily mono-neuronal but substantial changes in the receptrors occurs. It is during the early/intermediate stage that pharmacological and genetic prevention of PKC action blocks synapse elimination. Genetically modified (PKC theta knockout) animals show a delay of synapse loss developmentally but eventually the muscle innervation becomes completely mononeuronal. The use of PKC blockers over this same period in vivo had also completely prevented synapse elimination. We have tested what aspect of the neural activation process might be involved in the kinase mediated change in synapse effectiveness. Carbachol can be used to provide cholinergic stimulation of skeletal muscle and we have shown that such treatment of cultured skeletal myotubes results in activation and membrane translocation of PKC theta. A variety of experiments have implicated PKA as playing a crucial role in the stabilization of activated synapses. In view of the redundant mechanisms underlying activity-dependent synapse elimination implied by the studies of PKC block or knockout, we were interested in alternative messengers or mediators of the elimination/stabilization. In collaboration with Dr. Douglas Fields of the LCMN, NICHD, we have examined the possibility that ATP might be involved in the process. For this purpose we have used apyrase, an enzyme that breaks down ATP and suramin, a blocker of the P3 purinergic receptror that mediates some of the effects of ATP. In the presence of these agents, stimulation produces a significant decrement in synapse strength (stimulation of control, untreated preparations does not produce a decrement of the stimulated inputs). The depression lasts for over an hour after the drugs are washed out and stimulation of the preparation terminated. Large amounts of ATP are released from both nerve and muscle in our preparations and the release is modulated by activation of the nerve or muscle. Thus it seems possible that ATP may play a role in the stabilization of activated synapses, possibly through activation of a purinergic receptor. Activation of the PI3 kinase by wortmannin does not affect synapse synapse stability in either resting or stimulated synapses so that phosphorylations in general are not involved in synapse modulation. It seemed worthwhile to explore further the possibility that kinases other than PKA and PKC were involved in activity-dependent synapse modulation. The ras-mitogen-activated protein kinase (MAPK) and cAMP/PKA-mediated pathways play important roles in neuronal plasticity. We have used different blockers of the MAPK to test for its involvement in synapse modification in our system. Two such blockers are PD98059 and UO128. Neither of these produce synapse loss in the absence of synapse activation, but both, in conjunction with stimulation do produce loss. The inactive analogue of UO128, UO124, in the presence of synapse stimulation produces no synapse loss. These results suggest that MAPK may be involved in the Hebbian synapse modulation that occurs in our model system. Molecular bases for neurodevelopmental disorders We have utilized a variety of immunoaffinity analytical methods to determine the levels of several neuroactive molecules in blood samples from controls and children with different neurodevelopmental disorders. Through collaboration with members of the California Department of Health Services, we have access to blood specimens that were drawn at birth from a large sample of children and archived with subsequent diagnosis of some of the children as having autism or other developmental disorders. We have found that in very low birth weight children with and without diagnoses of infection or cerebral palsy that there was no correlation of clinical condition with levels of a number of cytokines. This was in contrast to significant differences in cytokine levels previously found to correlate with clinical condition in term infants. We have measured the neonatal blood levels of neurotrophins NT-3, NT-4 and Brain Derived Neurotrophic Factor (BDNF) and several cytokines in controls and in children subsequently shown to be autistic. There were so significant differences between cases and controls for IL-1 or IL-8, but the level of BDNF was about 25% lower in samples for autistic cases than in normals (p=0.032). This result was obtained with the double antibody sandwich immunoaffinity method used with the bead flow based technique of the Luminex system. It is different from the increase in BDNF (as well as some other analytes) found earlier with the single antibody recycling immunoaffinity chromatographic method. We are attemptiing to develop a single antibody version of the Luminex method to see if methodological issues explain this discrepancy.