In 1997, Zhu et al. established NF-L–null mice, which had no axonal NFs, reduced axonal radial growth, and delayed nerve regeneration. These results supported the long-standing view that NFs are the major determinant of axonal caliber, which is influenced in part by the cross-sectional density of NFs. These mice also had dramatically reduced levels of NF-M and NF-H (only ~5% of control level). Overexpression of NF-L alone does not increase the radial growth of axons (Xu et al., 1996
), but mice with larger axonal calibers were obtained by cooverexpression of either NF-L/NF-M or NF-L/NF-H, suggesting that NF-L is a key player, but other factors, such as the stoichiometric proportion of each subunit protein, may also be important in determining axonal diameter.
To help decipher the function of the NF-M and NF-H subunits, Elder et al. (1998a)
made null mutant mice lacking NF-M. These mice did not have any overt phenotypes or behavioral abnormalities. As noted above, however, the axonal caliber was dramatically decreased to ~50% of that in control littermates (Table ). Once again, though, interpretation of these results was difficult because the NF-M–null mice also had a drastic reduction in NF-L mRNA and protein levels. Tu et al. (1995)
also reported that overexpression of NF-M resulted in a corresponding increment in NF-L mRNA levels. These data and the aforementioned results of Zhu et al. (1997)
indicate that the levels of NF-L and NF-M are mutually regulated, reinforcing the idea that the stoichiometry of each NF subunit has an important role in determining axonal morphogenesis. In addition, the importance of NF-M in establishing NF-L assembly (Nakagawa et al., 1995
; Tu et al., 1995
) was also substantiated by this work.
Comparison of the Major Parameters among NF-M– and -H–targeting Mice
The three reports describing NF-H–null mice indicate that this subunit is less important than NF-M for NF assembly and structure. Although all three groups used essentially that same experimental procedures for establishing their mutant mice, we were amazed at the striking differences in morphological phenotype in samples taken from the same region at the same age. Here we will discuss these results in three contexts: (a) expression levels of mRNA and protein, (b) morphometric analysis (axonal caliber, nearest neighbor distance, NF density, MT density), and (c) specific experiments in each paper.
All three reports quantitated the expression level of NF-L and NF-M mRNA, because previous studies had suggested an interrelationship between subunit transcription levels (Tu et al., 1995
; Zhu et al., 1997
). In this case, however, the effect of NF-H ablation on the mRNA levels for NF-L and NF-M was negligible, suggesting that NF-H expression is relatively independent of the expression of the other two NF subunits, at least at the transcriptional level. What about posttranslational regulation? There are discrepancies in NF-M protein levels among these investigations, whereas all three report that NF-L protein levels are almost identical to wild type. Two studies report mild (~20%, Zhu et al., 1998
) to strong (~200%, Rao et al., 1998
) upregulation of NF-M, especially the phosphorylated form. This phenomenon could be explained in part by somewhat redundant roles of the COOH-terminal regions of NF-M and NF-H, which are the major phosphorylation sites. However, Elder et al. (1998b)
found normal levels of NF-M in the same preparation of tissue. It is difficult to rationalize this discrepancy, although it is possible that the later sampling time and the different tissue source in the latter study may be involved.
Rao and colleagues state that the axons in NF-H knockout mice resemble those in neurons undergoing axonal regeneration. In regenerating neurons, NF levels are downregulated while tubulin expression is highly upregulated (Hoffman et al., 1987
), both of which were observed in the NF-H null mutant by Rao et al. and Zhu et al. (NF levels are reduced to ~60% of normal and MT density is increased ~2.4-fold.) Since it has been suggested that the dephosphorylated COOH-terminal region of NF-H interacts with MTs, the increase in tubulin expression and elevated MT density may be a compensatory reaction for the stabilization of the axonal cytoskeleton. The slightly elevated expression level of tau in the NF-H null mutant (Zhu et al., 1998
) could also support this hypothesis because tau and the NF-H tail compete for binding sites on MTs (Miyasaka et al., 1993
). Alternatively, as Zhu et al. (1998)
suggest, increased MT density within axons may be due to accelerated transport in the absence of NFs.
Whereas the effects of NF-H ablation on the expression of the other two NF subunits were relatively predictable, the effects on the axonal caliber challenge the previous hypothesis that NF-H has a role in determining axonal diameter (deWaegh et al., 1992; Nixon et al., 1994
; Wong et al., 1995
). Zhu et al. (1998)
examined the L5 root axons containing both small- and large-caliber axons and found no significant difference between NF-H–null mutants and wild-type littermates, although a slight decrease (up to 10%) was observed in the large-caliber axons. This tendency was confirmed by Rao et al. (1998)
, who noted that only the largest caliber axon group in sensory neurons was affected. In this case, the effect may be due to the disruption of the cross-linking of NFs and actin by BPAG1n/dystonin, which is thought to be particularly important for the survival of sensory neurons (Yang et al., 1996
). Although none of the three groups determined the expression level of BPAG1n/dystonin, Rao et al. (1998)
quantitated the level of another cross-linker protein, plectin, which was not altered in mutant mice.
In contrast to the observations of the other two groups, the calibers of axons in the same tissue preparation were significantly reduced in the null mutant mice made by Elder et al. This discrepancy could be explained in part by the chronological differences between the data (Zhu et al., 1998
, and Rao et al., 1998
: 3 mo; Elder et al., 1998b
: 4 mo). Thus, the effect of deleting NF-H may emerge later in large caliber axons, or large caliber axons start to die in the mutant mice 3 wk postnatally, when the expression of NF-H is dramatically elevated in normal mice (Shaw and Weber, 1982
). To decipher the function of NF-H, it is important to determine if this time difference is significant. In nonmutant mice, NF-H levels are only ~20% of the adult level at 3 mo, whereas NF-L and NF-M levels are 60 and 90%, respectively. In fact, Rao et al. (1998)
described an overall reduction in the distribution of axonal diameters in 9-wk-old mutant mice, implying that NF-H may be significant for survival of the largest caliber neurons.
All three groups also examined another index of morphometry, the nearest neighbor distance, to ascertain whether NF-H exerts its effect on interfilament spacing, which might in turn determine the axonal caliber. No significant change in modal nearest neighbor distance was detected, which may indicate that the NF-H tail works redundantly with NF-M to determine the spacing between NFs.
Zhu et al. (1998)
extended their work to the neuropathology of NFs, which may help elucidate the mechanisms of neurodegenerative diseases. They treated both wild-type and NF-H–null mice with β,β′-iminodipropionitrile (IDPN), which is reported to segregate MTs from NFs, causing an accumulation of NFs within axons. This effect was not observed in NF-H–null mutant mice, reinforcing the hypothesis that NF-H participates in IDPN-induced neuropathy. These data might well serve as a toehold for extending these studies to the etiology of neurodegenerative disease, although the mechanism of NF-H involvement in this process is far from clear from this study.