Phosphorylation of highly conserved neurofilament medium KSP repeats is not required for myelin-dependent radial axonal growth

Abstract

Neurofilament medium (NF-M) is essential for the acquisition of normal axonal caliber in response to a myelin-dependent "outside-in" trigger for radial axonal growth. Removal of the tail domain and lysine-serine-proline (KSP) repeats of NF-M, but not neurofilament heavy, produced axons with impaired radial growth and reduced conduction velocities. These earlier findings supported myelin-dependent phosphorylation of NF-M KSP repeats as an essential component of axonal growth. As a direct test of whether phosphorylation of NF-M KSP repeats is the target for the myelin-derived signal, gene replacement has now been used to produce mice in which all serines of NF-M's KSP repeats have been replaced with phosphorylation-incompetent alanines. This substitution did not alter accumulation of the neurofilaments or their subunits. Axonal caliber and motor neuron conduction velocity of mice expressing KSP phospho-incompetent NF-M were also indistinguishable from wild-type mice. Thus, phosphorylation of NF-M KSP repeats is not an essential component for the acquisition of normal axonal caliber mediated by myelin-dependent outside-in signaling.

Figure 1.

Generation of a KSP phospho-incompetent NF-M mouse by substituting all KSP serine residues with alanine (NF-M^S→A). A, Mouse NF-M tail domain. The conserved sequence “KLLEGEE” (light blue) marks the end of the rod domain for all three neurofilament subunits. All KSP, KXSP, and KXXSP motifs are highlighted in bold, capital letters, as well as the variant KSD. The six previously identified highly phosphorylated KSP (Xu et al., 1992) motifs are indicated in red, and the nonphosphorylated KSP is indicated in dark blue. NF-M NCBI accession number NP_032717. B, Construction of an NF-M^S→A allele in which the serine residues of the highly conserved KSP repeats were mutated to alanine residues. The three exons of the NF-M gene are indicated by the filled boxes interrupted by two introns. ATG denotes the translation initiation codon. Dotted lines indicate the two regions where homologous recombination could take place between the targeting vector and the endogenous NF-M allele. C, Mouse genomic DNA was screened for targeting of the serine to alanine NF-M mutant using three primers for PCR-based genotyping. Top, A schematic representation of the endogenous NF-M gene depicts the approximate location of primer annealing sites of two of the three PCR primers with the predicted size of the amplified region of NF-M. Middle, A schematic representation of the targeted NF-M gene depicts the approximate location of primer annealing sites for all of the primers. The predicted size for the smaller amplified region is indicated. Bottom, Genotyping the NF-M loci by PCR of genomic DNA isolated from mouse-tail biopsies.

Figure 2.

Substitution of NF-M^S→A for wild-type NF-M does not affect stoichiometry of the neurofilament subunits. Expression of phospho-KSP-incompetent NF-M 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 6-month-old wild-type, NF-M^wt/S→A, NF-M^S→A/S→A, and NF-M^tailΔ homozygous mice were fractionated on 7.5% SDS polyacrylamide gels and stained with Coomassie blue (A) or immunoblotted (B) with antibodies that recognize NF-H in a phospho-independent manner (pAb-NF-HCOOH), NF-H in a phospho-dependent manner (RT-97 and SMI31), an epitope within the helical rod of NF-M (RMO-44), NF-L (NR-4), the neuron-specific βIII-tubulin (T8660), and β-tubulin (18D6).

Figure 3.

Absence of NF-M phosphorylation does not affect radial growth or survival of motor axons. A, Cross sections of the fifth lumbar motor (ventral) root from wild-type and NF-M^S→A homozygous mice at 2 months (left) or 6 months (right) of age. Scale bar, 10 μm. B, Number of axons in the fifth lumbar motor roots of 2- or 6-month-old wild-type and NF-M^S→A homozygous mice. Counts are average from four to five animals for each genotype. Means, for total axon counts, were analyzed by unpaired Student's t test. *p < 0.03. C, D, Distributions of axonal diameters in motor axons in 2-month-old (C) or 6-month-old (D) wild-type and NF-M^S→A homozygous mice. Each point represents the averaged distribution of axon diameters from the entire roots of five mice for each genotype and age group. Axonal populations were analyzed for overall statistical differences using a Mann–Whitney U test. There was a small, but statistically significant, difference between diameter distributions of wild-type versus NF-M^S→A mice at 2 months (p < 0.05). Error bars indicate SEM.

Figure 4.

Structure of axoplasm is slightly altered due to the expression of KSP phospho-incompetent NF-M. A, B, Transmission electron micrographs of 2-month-old motor axons derived from the fifth lumbar spinal cord segment of wild-type (A) and NF-M^S→A (B) homozygous mice. Scale bar, 400 nm. C, D, Distribution of nearest-neighbor distances from motor axons of 2-month-old (C) and 6-month-old (D) wild-type and NF-M^S→A homozygous mice. E, Neurofilament clustering, defined as the ratio of average filament spacing to nearest-neighbor spacing, was unaltered in wild-type versus NF-M^S→A mice at both 2 and 6 months, indicating that axoplasmic organization is similar in both mice. F–H, Quick-freeze, deep-etch micrographs of sciatic nerves from wild-type (F), NF-M^+/S→A (G), and NF-M^S→A/^S→A (H) mice. Scale bar, 400 nm. Arrowheads, Single, thin cross-linkers projecting from the core of neurofilaments. Arrows, Plectin-like linkers identified as more complex cross-linking structures projecting from the core of individual filaments. Many form Y or V structures. Error bars indicate SEM.

Figure 5.

Accumulation of cytoskeletal components is unaltered in motor neurons in mice expressing phospho-incompetent NF-M. A, The number of neurofilaments per axonal segment in both wild-type and NF-M^S→A homozygous mice at both 2 and 6 months. B, Microtubule content was reflective of overall axoplasmic organization. A trend toward accumulating more microtubules occurred in 2-month-old NF-M^S→A mice but did not reach statistical significance. This trend was not evident at 6 months, indicating no difference in axoplasmic organization. C, The ratio of microtubule number to neurofilament number was unchanged in wild-type versus NF-M^S→A mice. All data were analyzed for overall statistical analysis using ANOVA. Error bars indicate SEM.

Figure 6.

Expression of phospho-incompetent NF-M does not affect recovery rates from sciatic nerve injury. Functional recovery, by measuring the distance between the first and fifth digit, was measured in sciatic nerves that had been crushed at the level of the obturator tendon from wild-type and NF-M^S→A mice. Values were plotted as a percentage of motor function before crush injury. A minimum of five mice per genotype was assayed, and measurements were performed in triplicate for each animal per day for 21 d after crush injury. Error bars indicate SEM.