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. 2003 Dec 8;163(5):1011-20.
doi: 10.1083/jcb.200308159.

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Michael L Garcia  1 , Christian S Lobsiger, Sameer B Shah, Tom J Deerinck, John Crum, Darren Young, Christopher M Ward, Thomas O Crawford, Takahiro Gotow, Yasuo Uchiyama, Mark H Ellisman, Nigel A Calcutt, Don W Cleveland
Affiliations

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Michael L Garcia et al. J Cell Biol. .

Abstract

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

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Figures

Figure 1.
Figure 1.
Substitution of NF-Mtail Δ for wild-type NF-M does not affect stoichiometry of the neurofilament subunits. (A) Schematic of targeted NF-M gene in which 426 amino acids of the tail domain are replaced with a 12–amino acid c-myc epitope tag. The three exons of the NF-M gene are indicated by the filled boxes interrupted by three introns. ATG denotes the translation initiation codon. (B) Representation of NF-MtailΔ and NF-HtailΔ proteins generated by gene replacement of both endogenous NF-M and NF-H genes. (C) Mouse genomic DNA was screened for targeted truncation of NF-M and NF-H tails. (Left) Genotyping the NF-H loci by hybridizing with a sequence from NF-H (Rao et al., 2002). (Right) Genotyping the NF-M loci by hybridization to the sequence in A marked “5′ probe.” (D) Loss of NF-M, NF-H, or both tails has no effect on relative stoichiometries and accumulated levels of NF-L, NF-M, NF-H, or tubulin. Parallel immunoblots of sciatic nerve extracts from each of three sets of 2- and 6-mo-old wild-type, NF-HtailΔ, NF-MtailΔ, and NF-(M/H)tailΔ homozygous mice were fractionated on 7.5% SDS-polyacrylamide gels and stained with (top) Coomassie blue or immunoblotted with antibodies that recognize NF-H in a phospho-independent manner (pAb-NF-HCOOH), an epitope in the helical rod of NF-M (RMO-44), the epitope tag on NF-HtailΔ or NF-MtailΔ, NF-L, or the neuron-specific βIII-tubulin (MLR11).
Figure 2.
Figure 2.
Absence of NF-M or both NF-M and NF-H tail domains reduces radial growth of motor axons but does not markedly affect their survival. (A) Cross sections of L5 motor (ventral root) axonal profiles from wild-type, NF-MtailΔ, and NF-(M/H)tailΔ homozygous mice at (top) 2 mo or (bottom) 6 mo of age. Bar, 10 μm. Numbers of axons in L5 motor roots of 2- or 6-mo-old wild-type and NF-MtailΔ and NF-(M/H)tailΔ homozygous mice (B). Counts are average from four to five animals for each genotype. (C and D) Distributions of axonal diameters in motor axons in (C) 2- or (D) 6-mo-old wild-type, NF-HtailΔ, NF-MtailΔ, and NF-(M/H)tailΔ homozygous animals. Points represent the averaged distribution of axon diameters form the entire roots of five mice for each genotype and age group.
Figure 3.
Figure 3.
Structure of axoplasm in the presence or absence of NF-M and NF-H tail domains. (A–C) Transmission electron micrographs of 2-mo-old motor axons derived from the fifth lumbar spinal cord segment of (A) wild-type, (B) NF-MtailΔ, and (C) NF-(M/H)tailΔ mice. Bar, 200 nm. Distribution of nearest neighbor distances from motor axons of (D) 2- and (E) 6-mo-old wild-type, NF-MtailΔ, and NF-(M/H)tailΔ mice. (F–H) Quick-freeze, deep-etch micrographs of sciatic nerves from (F) wild-type, (G) NF-MtailΔ, and (H) NF-(M/H)tailΔ mice. Bar, 100 nm. Arrowheads point to cross-linkers projecting from the core of neurofilaments. Arrows point to plectin-like linkers.
Figure 4.
Figure 4.
Neurofilament tail domains are required for organizing axoplasm. (A) Neurofilaments (black dots) in cross sections of ideal, wild-type, and NF-(M/H)tailΔ axons were rearranged to form an array of regular hexagons encompassing the equivalent cross-sectional area. Note that the number of neurofilaments in all conditions is equal, however, the average distance each neurofilament is shifted to form triangle vertices is much greater for the tailless axon. (B) Neurofilament clustering, defined as the ratio of average filament spacing to nearest neighbor spacing, was significantly higher in both NF-MtailΔ and NF-(M/H)tailΔ mice at both 2 and 6 mo, indicating less axoplasmic organization in mice expressing truncated neurofilaments. Clustering was analyzed for overall statistical analysis using ANOVA with subsequent Tukey-Kramer multiple comparison post-hoc analysis for pairwise comparisons. *, P < 0.05 NF-MtailΔ versus wild type at 2 and 6 mo; **, P < 0.01 NF-(M/H)tailΔ versus wild type at 2 mo; ***, P < 0.001 NF-(M/H)tailΔ versus wild type at 6 mo. (C–E) Microtubule content was reflective of overall axoplasmic disorganization. A trend toward accumulating more microtubules occurred in both NF-MtailΔ and NF-(M/H)tailΔ mice (C) but did not reach statistical significance. (D and E) Electron micrographs from two different axons of the same L5 motor root from a single NF-(M/H)tailΔ mouse highlights the heterogeneity of microtubule accumulation that results from loss of both NF-M and NF-H tail domains. Bar, 200 nm. Microtubule content was analyzed for overall statistical significance using ANOVA with subsequent Bonferroni multiple comparison post-hoc analysis for pairwise comparisons. *, P < 0.05 NF-MtailΔ versus wild type. (F) Mitochondria accumulate in regions of high axoplasmic disorganization. At 6 mo, in both NF-MtailΔ and NF-(M/H)tailΔ mice, vesicle accumulation was significantly higher than age-matched controls (F). Vesicle accumulation was analyzed for statistical significance using Mann-Whitney test. *, P < 0.008 NF-MtailΔ versus wild type; **, P < 0.05 NF-(M/H)tailΔ versus wild type.
Figure 5.
Figure 5.
Absence of tail domains of NF-M or NF-M and NF-H slows nerve conduction velocity, but does not result in altered locomotor activity levels or recovery rates from sciatic nerve crush injury. (A) Nerve conduction velocity was measured from motor axons of the sciatic nerve from wild-type, NF-HtailΔ, NF-MtailΔ, and NF-(M/H)tailΔ mice. Values shown are from a minimum of five animals for each genotype, and the recordings are done in triplicate for each animal. Conduction velocities were analyzed for overall statistical analysis using ANOVA with subsequent Bonferroni multiple comparison post-hoc analysis for pairwise comparisons. *, P < 0.001 for NF-MtailΔ and NF-(M/H)tailΔ versus both wild type and NF-HtailΔ. (B) Average daily activity levels were analyzed in wild-type, NF-HtailΔ, NF-MtailΔ, and NF-(M/H)tailΔ mice. Mice were placed in activity chambers for a period of 14 d. Revolutions were stored on an activity wheel counter and then converted in kilometers based on a 5-inch diameter running wheel. A minimum of nine animals were analyzed for wild-type, NF-MtailΔ, and NF-(M/H)tailΔ mice, and four NF-HtailΔ mice were analyzed. (C) Functional recovery was measured in sciatic nerves that had been crushed at the level of the obturator tendon from wild-type, NF-HtailΔ, NF-MtailΔ, and NF-(M/H)tailΔ mice. Values were plotted as a percentage of motor function before crush injury. A minimum of five mice per genotype were assayed, and measurements were performed in triplicate for each animal per day for 21 d after crush injury.
Figure 6.
Figure 6.
Model of myelin-dependent outside-in signaling cascade that controls radial axonal growth. In normal axons, axoplasmic organization is dependent upon myelinating cells. Signal cascades (Blue Box) originating from MAG in the membrane of myelinating cells utilize the low affinity nerve growth factor receptor (p75NTR) in combination with neuronal gangliosides to transduce the myelin-dependent signal into the axoplasm. Upon association of MAG with p75NTR, members of the melanoma antigen (MAGE) gene family associate with intracellular domain of p75NTR, providing an intracellular scaffold for activation of ERK1/2 cascade, resulting in phosphorylation of NF-M (orange arrows) and NF-H (purple arrows) tail domains. MAG-dependent inactivation of p35/cdk-5 may reduce cdk-5 phosphorylation of NF-M and NF-H as well as prevent cdk-5–dependent inhibition of ERK1/2, allowing for near stoichiometric phosphorylation of NF-H and NF-M (yellow box). NF-M phosphorylation is required for establishing a volume-determining three-dimensional array (pink box) by a series of linkages that span between adjacent neurofilaments (green) and between neurofilament and microtubules (pink) or cortical actin (blue) filaments. Neurofilaments, microtubules, and cortical actin are interlinked by plakin family members of cytoskeletal linkers (blue) (Wiche, 1989; Eyer and Peterson, 1994; Svitkina et al., 1996). Axonal volume is, in part, established by a new class of linking protein that associates with neurofilaments in an NF-M tail phosphorylation–dependent manner to assist in long-range interactions that are necessary for the more than fivefold increase in axonal volume associated with radial growth.
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