Mechanisms of Neurodevelopment in health and disease

我们正在体外和体内研究在发育过程中激活对突触结构和功能的影响。活动依赖性突触消除是决定神经系统性能的主要过程，我们提出的证据表明，突触上的受体或其他分子受到不同蛋白激酶的作用，是这一过程的关键步骤。我们试图将我们的研究扩展到一些神经发育障碍，如自闭症。1)神经肌肉连接处活动依赖性突触丢失和稳定（Nelson, Jia, Li, Yang Lanuza, Valoria, Gizaw）。我们利用隔室组织培养系统（图1）来分析参与活动依赖性突触调节的一些过程的细胞轨迹。两个物理上分离的胆碱能神经元群（在系统的侧室）聚集在单个肌纤维群上。这些肌肉纤维的一个亚群受到来自两个神经元群的轴突的支配。对其中一组轴突的刺激会降低另一组的突触的突触效能，而在受两组神经细胞支配的肌肉细胞中，未受刺激的神经元群。先前的研究表明，蛋白激酶C （PKC）的作用是表达这种Hebbian，活性依赖性突触修饰所必需的。这项工作涉及PKC的药理激活和阻断，以及使用PKC θ异构体被敲除的动物细胞。由于肌肉细胞和神经元是分开制备和镀的，我们能够通过使用异种制备来评估神经元或肌肉细胞选择性丢失PKC的影响，其中神经或肌肉缺乏PKC，而另一种细胞类型是正常的。我们有证据表明突触效能的下调涉及突触后的变化，包括乙酰胆碱受体（AChR）的丧失。因此，正如我们所料，当正常神经支配的肌肉缺乏PKC时，我们发现没有活动依赖的突触调节。更令人惊讶的是，当PKC θ缺陷神经支配正常肌肉时，这种调节也不表达。电刺激或应用磷酸酯PMA激活PKC诱导的调节都是如此。此外，神经元PKC θ缺乏的影响本身依赖于活动。在这样的制备中，在神经元成分中缺乏PKC，并且用Na+通道阻滞剂河豚毒素电沉默，PMA确实产生突触下调。我们根据图2所示的模型解释了这些相当复杂的发现，以及蛋白激酶A （PKA）激动剂和拮抗剂的其他发现。PKA和PKC在突触的突触前和突触后组分中都起拮抗作用。PKA的作用对于维持神经末梢的递质输出和稳定突触后受体是必要的。相比之下，PKC阻止受体稳定物质的释放，可能是一种肽（如降钙素基因相关肽或CGRP）从神经释放，并在肌肉中起作用以破坏AChR的稳定。来自文献的大量证据表明，这种磷酸化模型可能是中枢神经系统活动依赖性突触可塑性的一些例子的基础。2)神经肌肉连接处GDNF的自分泌功能（Yang, Nelson）神经胶质源性神经营养因子（Glia Derived Neurotrophic Factor， GDNF）是一种由肌肉产生的营养因子，对脊髓运动神经元和整个大脑的其他神经细胞具有强大的作用。我们已经研究了GDNF也可能对肌肉细胞本身产生影响的可能性，特别是对乙酰胆碱受体（AChR）的代谢和膜定位。我们发现GDNF确实增加了肌细胞表面膜上AChR的插入率，对膜上的受体损失影响较小，对受体合成没有任何明显的影响。许多抑制剂研究表明，GDNF的作用是由α - 1 GDNF受体介导的，涉及Ret受体、MAP激酶、cAMP/CREB和Src激酶活性。因此，这种营养因子可能以突触前和突触后的协同方式起作用，以改变突触的功效。3)神经发育障碍的分子基础（Nelson, Satyanarayana, Song, Kuddo, VanDunk，与J. Grether和K.B. Nelson合作）我们可以访问正常儿童和随后被发现患有自闭症的儿童的存档新生儿血点。可获得的样品体积小，一些感兴趣的分析物含量低，而从血斑中洗脱出来的物质中包括血红蛋白在内的蛋白质含量高，这造成了相当大的技术问题。我们发现血液洗脱液中存在高水平干扰脑源性神经营养因子（BDNF）测定的物质。结合使用测定缓冲液稀释样品和使用已知的分析物峰值，我们表明来自自闭症儿童和对照组儿童的样品在BDNF水平或干扰物质水平上没有差异。同样，在我们的样本中，IL-1、IL-4、IL-8、TNF α和VEGF的水平也不能区分病例和对照组。合作者Judy Grether博士加州卫生服务部，Berkeley, California Karin B. Nelson, m.d.，神经流行病学分部，NINDS Terry Phillips博士，生物工程和物理科学部，NIH, Bethesda， MD。

We are studying the effect of activation upon synapse structure and function during development in vitro and in vivo. Activity-dependent synapse elimination is a major process determining nervous system performance and we present evidence that receptors or other molecules at the synapse are acted on by different protein kinases as a critical step in this process. We seek to extend our studies to some neurodevelopmental disorders such as autism. 1) Activity-dependent synapse loss and stabilization at the neuromuscular junction (Nelson, Jia, Li, Yang Lanuza, Valoria, Gizaw). We have utilized a compartmental tissue culture system (Fig. 1) to analyze the cellular locus of some of the processes involved in activity-dependent synapse modulation. Two physically separate populations of cholinergic neurons (in the side compartments of the system) converge on single population of muscle fibers. A subpopulation of these muscle fibers become innervated by axons from both of the neuronal populations. Stimulation of one set of axons produces down regulation of the synaptic efficacy of the synapses from the other, non-stimulated neuronal population in those muscle cells innervated by both populations. Previous work had shown that Protein Kinase C (PKC) action was required for expression of this Hebbian, activity-dependent synapse modification. This work involved pharmacological activation and blockade of PKC and the use of cells from animals in which the PKC theta isoform had been knocked out. Since the muscle cells and neurons are prepared and plated separately, we were able to assess the effect of selective loss of PKC from neurons or muscle cells by using heterologous preparations in which either nerve or muscle lacked the PKC while the other cell type was normal. We had evidence that the down-regulation of synapse efficacy involved post-synaptic changes including loss of acetylcholine receptors (AChR). As we therefore expected, when normal nerve innervated muscle lacking PKC theta, we found no activity-dependent synapse modulation. More surprisingly, the modulation was also not expressed when PKC theta deficient nerve innervated normal muscle. This was true with regulation induced by either electrical stimulation or PKC activation by application of the phorbol ester PMA. Furthermore, this effect of neuronal PKC theta deficiency was itself activity dependent. In such preparations that were lacking PKC theta in the neuronal component and that were electrically silenced with the Na+ channel blocker, tetrodotoxin, PMA did produce synapse down regulation. We interpret these rather complex findings, and other findings with Protein Kinase A (PKA) agonists and antagonists, in terms of the model shown in Fig. 2. PKA and PKC play antagonistic roles in both pre- and post-synaptic components of the synapse. PKA actions are necessary to sustain transmitter output from the nerve terminal and to stabilize the postsynaptic receptor. PKC, by contrast, blocks release of receptor stabilizing material, perhaps a peptide (such as Calcitonin Gene Related Peptide or CGRP) from the nerve and acts in the muscle to de-stabilize the AChR. Considerable evidence from the literature suggest that this phosphorylation model may underlie some examples of activity-dependent synaptic plasticity in the central nervous system.2) Autocrine function for GDNF at the neuromuscular junction (Yang, Nelson) Glia Derived Neurotrophic Factor (GDNF) is known to be a trophin produced by muscle with powerful effects on spinal motor neurons and other nerve cells throughout the brain. We have examined the possibility that GDNF could also have an effect on the muscle cell itself, in particular on the metabolism and membrane localization of the acetylcholine receptror (AChR). We find that GDNF does increase the insertion rate of AChR into the surface membrane of the muscle cells, with a lesser effect on receptor loss from the membrane and without having any appreciable effect on receptor synthesis. A number of 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, this trophin may act in a synergistic pre-and postsynaptic manner to modify synapse efficacy. 3) Molecular basis for neurodevelopomental disorders (Nelson, Satyanarayana, Song, Kuddo, VanDunk, in collaboration with J. Grether and K.B. Nelson) We have access to archived neonatal blood spots from normal children and children subsequently found to be autistic. The small volumes of the available samples, the low levels of some of the analytes of interest and the high levels of proteins including hemoglobin in the material eluted from the blood spots have posed considerable technical problems. We find that high levels of materials that interfere with the assay for Brain Derived Neurotrophic Factor (BDNF) are present in the blood eluates. With a combination of sample dilution with an assay buffer and the use of known analyte spikes we show that samples from autistic and control children do not differ either in the the levels of BDNF or in the levels of the interfering substances. Similarly, levels of IL-1, IL-4, IL-8, TNF alpha and VEGF do not distinguish cases and controls in our sample. Collaborators Judy Grether, Ph.D. California Department of Health Services, Berkeley, California Karin B. Nelson, M.D., Neuroepidemiology Branch, NINDS Terry Phillips Ph.D. Division of Bioengineering and Physical Science, NIH, Bethesda, MD.