NEUROFILAMENT DEPENDENT STRUCTURING OF AXOPLASM

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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.