NEUROFILAMENT DEPENDENT STRUCTURING OF AXOPLASM

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。原理与背景：神经丝是大髓鞘轴突中最丰富的结构成分，是轻（NF-L）、中（NF-M）和重（NF-H）神经丝的专性异质聚合物。与大多数神经元蛋白一样，神经丝在体细胞中合成，随后通过缓慢的轴突运输转运到轴突。一旦进入轴突，神经丝是非常长寿的蛋白质，参与建立和维持轴质的三维阵列。对压伤后轴突恢复后神经丝表达的分析表明，神经丝在建立轴突直径方面发挥了作用（Hoffman et al., 1987）。小鼠和鹌鹑的遗传学都明确证实，神经丝是决定成熟轴突直径所必需的。在小鼠实验中，神经丝的缺失（Elder et al., 1998; Eyer and Peterson, 1994; Jacomy et al., 1999; Ohara et al., 1993; Zhu et al., 1997）显著抑制髓鞘形成过程中开始的轴突直径的增长。此外，轴突直径对神经丝的亚基比例敏感，因为任何单个亚基的表达增加都会抑制径向生长（Collard等人，1995；Cote等人，1993；Marszalek等人，1996；Monteiro等人，1990；Tu等人，1995；Wong等人，1995；Xu等人，1996），而同时过表达NF-L和NF-M或NF-H会增加总轴突直径（Xu等人，1996）。神经丝依赖的径向生长本身与NF-M和NF-H的羧基末端尾部结构域的磷酸化有关。在Sf9昆虫细胞中，全长神经丝的表达和各种截断突变导致10 nm的纤维形成羧基末端，磷酸化的交叉桥沿着纤维长度延伸（Chen et al., 2000; Nakagawa et al., 1995）。对坐骨神经轴质的分析表明，这些羧基末端的交叉桥与相邻的神经丝和微管接触（Hirokawa et al., 1984; Rao et al., 2002）。此外，羧基末端截断的NF-H的表达导致小鼠径向生长速率降低，交叉桥明显减少。有趣的是，NF-H的羧基末端截断导致了在野生型幼崽中没有观察到的紧密对立的神经丝亚区（Rao et al., 2002）。虽然神经丝及其磷酸化是轴突出生后正常生长所必需的，但一些证据表明，神经丝磷酸化是由髓鞘细胞调节的（de Waegh et al., 1992; Yin et al., 1998）。髓磷脂缺陷的雪旺细胞所包裹的轴突片段不能在出生后生长，而髓磷脂正常的雪旺细胞所包裹的同一轴突片段可以获得较大的轴突直径（de Waegh et al., 1992）。这些数据有力地表明，起源于髓鞘细胞的“由外而内”信号级联激活了局部激酶或磷酸酶（或两者都有），导致NF-M和NF-H的羧基末端局部磷酸化增加，从而导致轴突扩张（Hsieh等，1994）。此外，位于节间的神经丝在NF-M和NF-H的羧基末端尾部结构域中几乎被化学计量磷酸化，而位于Ranvier节点的nf则被磷酸化得少得多（Carden et al., 1985; Julien and Mushynski, 1983; Lee et al., 1988）。基因靶向和细胞培养研究表明，NF-M可能是髓磷脂衍生信号的靶标，导致轴突径向生长（Elder et al., 1998）。然而，整个NF-M的缺失并不能让我们深入了解髓磷脂衍生信号级联靶向的NF-M的特定子区域。为了测试NF-M亚基尾部结构域的这种功能，我们现在通过胚胎干细胞介导的基因敲入方法构建了NF-M无尾小鼠。利用高压电子显微镜结合电子断层扫描，我们将分析整个成熟过程中神经丝阵列的精确三维组织。此外，我们将分析髓磷脂结构的改变，这可能是由于小鼠NF-M尾部结构域或小鼠NF-M和NF-H尾部结构域上所有假定的磷酸化位点的慢性丧失而导致的。目的：这些研究的目的是直接确定神经丝蛋白在成熟的哺乳动物周围神经系统的轴质结构中的作用。先前利用基因缺失的研究已经明确地确立了神经丝在确定成熟轴突口径建立过程中使用的神经丝阵列的精确三维结构中的作用，而成熟轴突口径的建立最终影响神经冲动传递的速率。为了确定轴突生长的结构原理，我们培养了表达羧基末端截断的NF-M、NF-H或两者都表达的小鼠，以及一个或多个神经丝亚基被系统删除的小鼠。通过使用高压电子显微镜和随后的形态计量学分析，我们确定了通过纤维间最近邻间距判断的纤维间组织不受NF-M或NF-H尾域损失的影响。此外，神经丝支架在横截面上没有明显改变。然而，同时删除两个尾结构域显著减少了纤维间的距离，并导致神经纤维支架缺乏相互连接，交叉桥结构。高压电子显微镜和电子断层扫描纵向切片现在设想，通过NCMIR资源，以确定在缺乏神经丝亚基和神经丝尾结构域的情况下功能性神经丝支架组织的改变，以及这些改变对神经系统成熟所需的轴质整体精确三维组织的影响。此外，神经的三维重建将允许评估轴浆结构的改变如何与快速冲动传递所需的神经功能域相关，例如髓鞘副神经环。高压电子显微镜和断层扫描重建对基因工程小鼠神经的应用，使得详细描述神经丝依赖的轴质结构和改变轴质组织的后果，以及在突触传递中至关重要的轴胶质相互作用的微观解剖成为可能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rationale and Background: Neurofilaments are the most abundant structural component of large myelinated axons, and are obligate hetero-polymers of neurofilament light (NF-L), medium (NF-M) and heavy (NF-H). As with most neuronal proteins, neurofilaments are synthesized in the soma, and are subsequently transported into the axon via slow axonal transport. Once in the axon, neurofilaments are extremely long-lived proteins involved in establishing and maintaining the three-dimensional array of axoplasm.Analysis of neurofilament expression following axonal recovery from crush injury suggested a role for neurofilaments in establishing axonal diameter (Hoffman et al., 1987). Genetics in both mouse and quail have unequivocally confirmed that neurofilaments are required for determining mature axonal diameter. In mouse, loss of neurofilaments (Elder et al., 1998; Eyer and Peterson, 1994; Jacomy et al., 1999; Ohara et al., 1993; Zhu et al., 1997) markedly suppresses the growth in axonal diameter that initiates during myelination. Moreover, axonal diameter is sensitive to the subunit ratio of neurofilaments as increased expression of any single subunit inhibits radial growth (Collard et al., 1995; Cote et al., 1993; Marszalek et al., 1996; Monteiro et al., 1990; Tu et al., 1995; Wong et al., 1995; Xu et al., 1996) whereas simultaneous overexpression of NF-L and NF-M or NF-H increases overall axonal diameter (Xu et al., 1996). Neurofilament dependent radial growth is itself associated with phosphorylation of the carboxy terminal tail domains of both NF-M and NF-H. Expression of full-length neurofilaments, and various truncation mutations, in Sf9 insect cells resulted in 10 nm fibers that formed carboxy terminal, phosphorylated cross-bridges that extended along the length of the filament (Chen et al., 2000; Nakagawa et al., 1995). Analysis of the sciatic nerve axoplasm suggested that these carboxy terminal cross-bridges contact adjacent neurofilaments and microtubules (Hirokawa et al., 1984; Rao et al., 2002). Furthermore, expression of carboxy terminally truncated NF-H resulted in mice with reduced rates of radial growth and markedly fewer cross-bridges. Interestingly, carboxy-terminal truncation of NF-H resulted in sub-regions of closely opposed neurofilaments that were not observed in wild type littermates (Rao et al., 2002). While neurofilaments and their phosphorylation are required for proper post-natal growth of axons, several lines of evidence suggests that neurofilament phosphorylation is regulated by myelinating cells (de Waegh et al., 1992; Yin et al., 1998). Axonal segments ensheathed by myelin defective Schwann cells do not undergo post-natal growth whereas segments of the same axon ensheathed by myelin-competent Schwann cells achieve large axonal diameters (de Waegh et al., 1992). These data strongly suggest that an ¿outside-in¿ signal cascade, originating from myelinating cells, activates local kinases or phosphotases (or both) resulting in increased local phosphorylation of the carboxy termini of NF-M and NF-H resulting in expansion of the axon (Hsieh et al., 1994). Moreover, neurofilaments that reside within the internode are nearly stoichoimetrically phosphorylated on the carboxy-terminal tail domain of NF-M and NF-H whereas NFs that reside in the Node of Ranvier are considerably less phosphorylated (Carden et al., 1985; Julien and Mushynski, 1983; Lee et al., 1988).Gene targeting and cell culture studies implicate NF-M as a possible target for a myelin-derived signal resulting in radial growth of axons (Elder et al., 1998). However, deletion of the entirety of NF-M does not offer insight into the specific sub-region(s) of NF-M targeted by a myelin-derived signal cascade. To test this proposed function of the NF-M subunit tail domain, we have now constructed NF-M tailless mice by embryonic stem cell mediated gene knock in approach. Utilizing high voltage electron microscopy in combination with electron tomography, we will analyze the precise three dimensional organization of the neurofilament array throughout maturation. Additionally, we will analyze alterations in myelin structure that may result as a consequence of chronic loss of all putative phosphorylation sites on either the murine NF-M tail domain or both murine NF-M and NF-H tail domains.Objectives: The goal of these studies is to directly ascertain the role(s) of neurofilament proteins utilized in structuring of axoplasm within the maturing, mammalian peripheral nervous system. Previous studies, utilizing gene deletion, have unequivocally established a role for neurofilaments in determining the precise three dimensional architecture of the neurofilament array utilized during establishment of mature axon caliber, a property which ultimately influences the rate of nerve impulse transmission. To identify the structural principles of axonal growth, we have generated mice that express either carboxy-terminally truncated NF-M, NF-H or both as well as mice which have had one or more neurofilament subunits systemically deleted. Through the use of high voltage electron microscopy with subsequent morphometeric analysis, we have determined that interfilament organization, judged by interfilament nearest neighbor spacing is unaffected by loss of either NF-M or NF-H tail domain. Additionally, the neurofilament scaffold does not appear significantly altered in cross section. However, simultaneous deletion of both tail domains significantly reduces interfilament distances, and results in a neurofilament scaffold that is devoid of interlinking, crossbridge structures. High voltage electron microscopy and electron tomography of longitudinal sections is now envisioned, through NCMIR resources, to identify the alterations to the organization of a functional neurofilament scaffold in the absence of neurofilament subunits and neurofilament tail domains as well as the consequences these alterations have to the overall precise three-dimensional organization of axoplasm required for maturation of the nervous system. Additionally, three dimensional reconstructions of nerves will allow assessment of how alterations in axoplasmic structuring correlated with nerve functional domains required for rapid impulse transmission, such as paranodal looping of myelin. The application of high voltage electron microscopy and tomographic reconstruction to nerves of genetically engineered mice makes it possible to detail neurofilament dependent axoplasmic structuring and consequences, of altering axoplasmic organization, to the microanatomy of axo-glial interactions that are of key importance in synaptic transmission.