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© The Rockefeller University Press,
0021-9525/2000//1013 $5.00
The Journal of Cell Biology, Volume 151, Number 5,
, 2000 1013-1024
Original Article |
Local Control of Neurofilament Accumulation during Radial Growth of Myelinating Axons in Vivo
: Selective Role of Site-Specific Phosphorylation
b Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115
c Laboratories for Molecular Neuroscience, McLean Hospital, Belmont, Massachusetts
d Ludwig Cancer Institute at University of California at San Diego, La Jolla, California 92093
The accumulation of neurofilaments required for postnatal radial growth of myelinated axons is controlled regionally along axons by oligodendroglia. Developmentally regulated processes previously suspected of modulating neurofilament number, including heavy neurofilament subunit (NFH) expression, attainment of mature neurofilament subunit stoichiometry, and expansion of interneurofilament spacing cannot be primary determinants of regional accumulation as we show each of these factors precede accumulation by days or weeks. Rather, we find that regional neurofilament accumulation is selectively associated with phosphorylation of a subset of Lys-Ser-Pro (KSP) motifs on heavy neurofilament subunits and medium-size neurofilament subunits (NFMs), rising >50-fold selectively in the expanding portions of optic axons. In mice deleted in NFH, substantial preservation of regional neurofilament accumulation was accompanied by increased levels of the same phosphorylated KSP epitope on NFM. Interruption of oligodendroglial signaling to axons in Shiverer mutant mice, which selectively inhibited this site-specific phosphorylation, reduced regional neurofilament accumulation without affecting other neurofilament properties or aspects of NFH phosphorylation. We conclude that phosphorylation of a specific KSP motif triggered by glia is a key aspect of the regulation of neurofilament number in axons during axonal radial growth.
Key Words: axon caliber • axon–glia interactions • oligodendroglia • CNS development • protein phosphorylation
© 2000 The Rockefeller University Press
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Introduction |
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Neurofilament number within axons is influenced by several factors. After axons make synaptic contacts, the synthesis of neurofilament subunits and other cytoskeletal proteins increases and the rates of slow axonal transport of these proteins decrease severalfold (Hoffman et al. 1983; Willard and Simon 1983). These changes may raise the levels of many cytoskeletal proteins along the entire length of the axon, but they do not account for the selective expansion of myelinating axonal regions, which achieve diameters and numbers of neurofilaments that may be manyfold larger than those in an immediately adjacent unmyelinated region. Developmentally regulated properties of neurofilaments that might mediate neurofilament accumulation selectively within a discrete region of an axon include the state of phosphorylation and subunit composition as considered briefly below.
During early postnatal development, the COOH-terminal side arm domains of NFH and NFM subunits become extensively phosphorylated within a multiphosphorylation repeat (MPR) region containing the sequence motif Lys-Ser-Pro (KSP) (Carden et al. 1987; Clark and Lee 1991). In the case of NFH, the >40 KSP repeats present in this region can be separated into two categories, KSPXX and KSPXK (where the final X is any amino acid except lysine), which are believed to be regulated by different protein kinases (Bennett et al. 1994; Elhanany et al. 1994). Several studies suggest that, among numerous candidate neurofilament protein kinases (115 kD, glycogen synthetic kinase [GSK] 3, extracellular signal–regulated kinase [ERK], stress-activated protein kinase, protein kinase K, protein kinase C), Cdk-5 preferentially phosphorylates the KSPXK repeats (Hisanaga et al. 1993; Shetty et al. 1993; Guidato et al. 1996; Sun et al. 1996) and has been reported to generate epitopes recognized by the monoclonal antibodies SMI31 and RT97 (Bajaj and Miller 1997). In contrast, ERKs and GSK appear to regulate KSPXX repeats and generate the SMI34 as well as SMI31 epitopes (Roder and Ingram 1991; Pant and Veeranna 1995). Phosphorylation at the COOH-terminal domains of NFH and NFM in vitro straightens individual neurofilaments and promotes their alignment into bundles (Leterrier et al. 1996) and, in vivo, is associated with an increased interneurofilament spacing (Hsieh et al. 1994; Nixon et al. 1994), as NFH and NFM COOH-terminal side arms extend and form crossbridges among neurofilaments and other cytoskeletal elements (Hirokawa et al. 1984; Gotow and Tanaka 1994; Gotow et al. 1994). Neurofilament transport slows when NFH and NFM are extensively phosphorylated (Watson et al. 1989; Archer et al. 1994). When these subunits are at their highest states of phosphorylation, neurofilaments may stop moving for extremely long periods of time (Lewis and Nixon 1988), presumably reflecting their integration within a stationary but dynamic cytoskeletal network along axons (for reviews see Hirokawa et al. 1984; Nixon 1998a,Nixon 1998b). Although it is likely that the different properties and behaviors of neurofilaments within axons are independently regulated by the phosphorylation of specific sites within the MPR domain, this possibility has not been previously addressed.
The subunit composition of neurofilaments also influences neurofilament number in axons under some conditions (Cote et al. 1993; Collard et al. 1995; Wong et al. 1995; Xu et al. 1996). The delayed expression of NFH during development occurs during the same period as when slow axonal transport rates decrease and neurofilament levels within axons increase (Hoffman et al. 1983; Willard and Simon 1983). Moreover, increasing the NFH content of neurofilaments by overexpressing NFH in transgenic mice slows neurofilament transport and raises axonal levels of neurofilaments proximally along axons (Cote et al. 1993; Collard et al. 1995; Marszalek et al. 1996). In both of these situations, the potential contribution of subunit phosphorylation to these effects is unclear. More recently, targeted disruption of neurofilament subunit genes (Elder et al. 1998a; Rao et al. 1998; Zhu et al. 1998) has been used to investigate the roles of each neurofilament subunit in axon radial growth. Ablation of the genes for either NFM or NFL significantly reduced neurofilament numbers and axonal calibers (Ohara et al. 1993; Zhu et al. 1997; Elder et al. 1998a). Neurofilament assembly requires NFL (Ching and Liem 1993; Lee et al. 1993), and the markedly lowered NFL levels in NFM-deleted mice (Elder et al. 1998b) presumably accounts in part for reduced axon calibers in these mice. Mice lacking NFH have also been generated (Elder et al. 1998b; Rao et al. 1998; Zhu et al. 1998). The small though appreciable effects of this gene deletion on motor axon caliber led some investigators to question the role of NFH in determining axon caliber and, by inference, regulating neurofilament number. However, revealing the function of a protein based solely on the magnitude of phenotypic changes after gene ablation may be problematic because functions of the deleted protein are often compensated by other proteins. In this regard, modest caliber reductions were observed in NFH-deleted mice, but effects of NFH deletion were partially compensated by increases in NFM levels and numbers of microtubules (Rao et al. 1998; Zhu et al. 1998).
To distinguish the roles of subunit stoichiometry and phosphorylation as determinants of regional neurofilament accumulation, we characterized the changes in molecular and structural properties of neurofilaments as they occurred differentially in expanding and unexpanded portions of mouse optic axons during development. We combined analyses of normal retinal ganglion cell development with specific perturbations of neurofilament behavior in NFH-deleted mice and in Shiverer (Shi) mutant mice carrying a mutation of the myelin basic protein (MBP) gene, which interferes with oligodendrocyte maturation (Chernoff 1981; Shine et al. 1992) and prevents most axons from receiving signals that trigger regional neurofilament accumulation (Sanchez et al. 1996). Our results show that the regional neurofilament accumulation associated with the greatest radial growth of axons begins well after neurofilament subunits achieve mature stoichiometry and, instead, is closely related to the phosphorylation of a subset of KSP sites on the NFH COOH terminus. In the absence of NFH, NFM partially assumes the role of this phosphorylation event during regional neurofilament accumulation.
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Electron Microscopy
Mice were anesthetized with halothane gas and the tissue was fixed through intracardial perfusion with 4% paraformaldehyde, 5% glutaraldehyde in 0.1 M PBS, pH 7.4, at room temperature. The optic nerve was dissected and processed as described by Nixon et al. 1994, segmented in 1.2-mm pieces, cleared in propylene oxide, and embedded in Medcast (Ted Pella, Inc.). Ultrathin sections were collected and the section containing the initial portion of the retinal excavation was then used as a standard reference point. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a EX electron microscope (model JEM1200; JEOL) at 80 kV.
Immunoelectron Microscopy
Thin sections of epon-embedded tissue were picked up on formvar-coated nickel grids. These sections were etched with 1% periodic acid for 10 min, rinsed in double distilled water (ddw), etched with 3% sodium metaperiodate for 30 min, and rinsed in ddw, followed by 0.05 M Tris-buffered saline (TBS), pH 7.6, for 5 min. Sections were then blocked with 20% normal goat serum in TBS for 60 min at room temperature and then incubated overnight at room temperature in a moist chamber with primary antibody (RT97) diluted at 1:5,000 in TBS containing 1% normal goat serum, 1% BSA, and 0.1% Triton X-100. The sections were washed sequentially in TBS, pH 7.6, for 5 min, in TBS, pH 8.2, for 5 min, and in TBS, pH 8.2, with 0.5% polyethylene glycol for 5 min. Sections were then incubated in 5-nm gold conjugate IgG diluted to 1:25 in TBS containing polyethylene glycol, pH 8.2, for 2 h at room temperature, followed by rinses, first in TBS then in ddw. Finally, the sections were counterstained in uranyl acetate and lead citrate and examined with an EX microscope (model JEM1200; JEOL) with an AMT digital camera.
Determination of Axonal Diameter Sizes and Quantitation of Neurofilaments
Axonal diameters were determined from 1,561–4,919 axons at the 50- and 700-µm levels from each of the six postnatal ages analyzed (P9, 12, 16, 21, 30, and 120) and from NFH-deleted and control mice aged 1.5–1.7 yr old. Neurofilament and microtubule numbers in each optic nerve were determined from electron micrographs at the 50- and 700-µm levels by counting 600–1,185 axon profiles representative of the range in calibers in the total axonal population to accurately reflect absolute numbers of neurofilaments in a population of axons heterogeneous in caliber. Values were expressed as number of neurofilaments per 1,000 axons as described previously (Sanchez et al. 1996).
SDS-PAGE, Immunoblotting, and Two-dimensional Gel Electrophoresis
The most proximal 2.2-mm segment of the optic nerve from C57Bl/6J mice at postnatal ages of 4 d through adult (120 d) was removed at 4°C after mice were killed by cervical dislocation. Segments from eight mice of a given age were pooled and stored at –70°C. The optic nerve cytoskeleton proteins were obtained from pooled segments for each given age. All subunit levels in developing mice were measured based on the standard optic nerve length. In studies of adult NFH-null and Shiverer mice, we used the standard 9-mm length of optic pathway used in previous studies (Nixon and Logvinenko 1986), which extended from the eye to the optic tract. The tissue was homogenized as shown by Chiu and Norton 1982 with 50 mM Tris-HCl, pH 6.8, buffer containing 1% Triton, 50 mM NaF, 5 mM NaVO3, 10 µm genistein, 0.25 M NaCl, 50 µg/ml leupeptin, 0.1% aprotinin, and 2 mM PMSF (buffer A). The homogenate was centrifuged at 15,000 g for 10 min at 4°C and the pellet and supernatant were separated and processed immediately. Equal aliquots of Triton-insoluble fractions pooled from the 2.2-mm proximal segments as described were loaded onto each lane. Protein separation was accomplished by gel electrophoresis, as described by Laemmli 1970, with the use of 10% polyacrylamide gels and a Hoefer minigel format for most experiments. Proteins were transferred to nitrocellulose membranes as described by Towbin et al. 1979, except for the inclusion of 0.1% SDS and the omission of methanol in the transfer buffer. Nonfat milk (5%) in TBS with 0.3% Tween was used to block nonspecific antibody binding. Total levels of NFH protein were determined using the antibody SMI33, which recognizes NFH and NFM, irrespective of their phosphorylation states. Varying states of phosphorylation on the COOH terminus of NFH were detected using the phosphorylation-dependent antibodies SMI31, SMI34, and RT97 (Sternberger and Sternberger 1983; Coleman and Anderton 1990). Immunostaining was performed using HRP-labeled anti–rabbit or anti–mouse biotinylated antibodies and a DAB chromagen as described by the manufacturer (Vector Laboratories). NFH levels in the Triton-insoluble and -soluble protein fractions were qualitatively determined by using the ECL chemiluminescence developing system (Amersham Pharmacia Biotech). Immunoreactivity levels were quantified by analyzing digitized images generated by the Ofoto program (Light Source, Inc.) with Scan Analysis® software (Specom Research). Quantitations were performed within the linear range of the densitomer. A standard curve for each antibody was derived from subsequent dilutions of adult neurofilament protein immunoblotted and stained with the respective antibodies. Immunoreactivity levels were expressed relative to the value at P120 for each antibody separately. The data were plotted using a linear scale on the x-axis. Two-dimensional electrophoresis (3.5–9 pH range) was performed according to O'Farrell 1975, using the Hoefer 460 system.
Confocal Immunofluorescence Microscopy
C57Bl/6J mice were anesthetized with haloethane gas and perfused intracardially with 4% paraformaldehyde in PBS, pH 7.4. Retina whole mounts were stained with a cocktail of N-52 (Sigma-Aldrich) and SMI32 (Sternberger Monoclonal) anti-NFH antibodies to recognize all forms of NFH. The immunoreactivity was visualized with a 410 LSM Laser Confocal system (ZEISS) after staining with fluorescein labeled anti–mouse antibody (Vector Laboratories).
Proteolytic Digestion and HPLC Separation of NFH Peptides
The mouse spinal cord cytoskeleton was prepared in the presence of phosphatase and protease inhibitors as described previously (Sihag and Nixon 1989) and the proteins were separated on 24-cm-long 6% SDS-polyacrylamide gels. The 200-kD polypeptide band identified as NFH was excised from gel, and the gel slices were washed extensively with 20% methanol. The gel slices were lyophilized and homogenized and the NFH polypeptide was digested with 2 µg/ml TPCK-trypsin or TLCK-
-chymotrypsin in 50 mM NH4HCO3 for 20 h at 37°C. The proteolytic reactions were inhibited by addition of 10 µg/ml of AEBSF, 10 µg/ml aprotinin, and 2 mM PMSF. The resulting NFH peptides were lyophilized, solubilized in a 20% acetic acid and 5% formic acid solution, and separated on a preparative C18 reverse-phase HPLC column as described previously (Sihag and Nixon 1989). The 2–80% acetonitrile gradient was run in 45 min and 1-ml/min fractions were collected. The HPLC fractions from 3 to 30 containing the tryptic or TLCK--chymotrypsin NFH peptides were lyophilized, solubilized in 50 mM NH4 HCO3, and either dot-blotted on a 0.22-µm PVDF membrane or pooled to be further affinity-purified, respectively. Immunoreactivity of the dot-blotted peptides was examined with SMI34, SMI31, or RT97.
Affinity Purification of RT97 and SMI31 Immunoreactive Peptides
Cyanobromide-activated Sepharose 4B (BD PharMingen) was coupled to protein A–purified RT97 or to SMI31 antibodies using the protocol recommended by the manufacturer, and the slurry was poured into 1-ml syringe columns. The tryptic and chymotryptic digests of NFH were prepared as described above. Equal aliquots of the digest were passed thrice through the RT97 or SMI31 affinity columns. The unbound peptides were removed by several washes with 10 volumes of TBS. The NFH peptides that bound to the RT97 or SMI31 affinity columns were eluted with 0.1 M glycine, pH 2.7, and neutralized with 1 M Tris-HCl, pH 8.0. The column fractions were then analyzed by dot blot on PVDF membranes. The immunoreactivity of the eluted peptides from each column was tested against both RT97 and SMI31 by using the ECL development system (Amersham Pharmacia Biotech).
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100 µm after they converge at the retinal excavation to form the optic nerve (Fig. 1A and Fig. B). Beyond the lamina cribrosa, which is a specialized meshwork of astrocytes located at 100–150 µm from the retinal excavation, >95% of the axons in adult mice are myelinated, larger in caliber, and contain more neurofilaments (Fig. 1A and Fig. C) (Nixon et al. 1994). During postnatal development, the cross-sectional areas of optic axons at levels beyond the lamina cribrosa increase an additional 200% under the control of signals from oligodendroglial cells (Sanchez et al. 1996). Our results showed that >80% of the total regional caliber growth occurred between 21 and 30 d postnatally (Fig. 1D and Fig. E). Regional caliber expansion is defined as the percent increase in the axonal cross-sectional area at the 700-µm level (or more distal levels) over the area at a level 50 µm (Fig. 1 E) from the retinal excavation (i.e., Area [700 µm – 50 µm/50 µm] x 100).
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Attainment of Mature Neurofilament Subunit Stoichiometry and Interneurofilament Spacing Precede Regional Neurofilament Accumulation and Caliber Expansion
To investigate how subunit stoichiometry contributes to regional neurofilament accumulation, the subunit composition of neurofilaments from optic nerves isolated at stages throughout postnatal development was quantitated by densitometric analysis of Western blots. For comparison, we analyzed isolated neurofilaments from the intraretinal portions of optic axons where neurofilaments undergo minimal reorganization (Sanchez et al. 1996). Although the retina is composed of different cell types, the axons and perikarya of retinal ganglion cells (RGCs) contained nearly all of the NFH immunoreactivity as shown by confocal microscopic analysis of whole mount preparations of retina immunostained with anti-NFH antibodies (data not shown). Analysis of the Triton-soluble NFH pool that has been proposed to incorporate into assembled neurofilaments during development (Shea 1994) revealed very little if any Triton-soluble NFH in the optic nerve after P10, although a pool of Triton-soluble NFH protein persisted in the retina throughout development (Fig. 2 A). NFH levels increased nearly threefold between P9 and P16 along optic axons, including intraretinal portions (Fig. 2 B). Consistent with these results, the ratio of NFH to NFL rose sharply from P6 and peaked by P12 along axons (Fig. 2 C), nearly 2 wk before neurofilaments began to accumulate regionally (Fig. 1 G) and well before mature axonal diameters were achieved (Fig. 1 E) (Sanchez et al. 1996). The proportions of NFM in optic axon neurofilaments decreased (Fig. 2 C) as the ratio of NFH to NFL increased. Comparing the subunit stoichiometries at different postnatal ages confirms a net reduction in NFM content on neurofilaments when NFH appears (Fig. 2 D).
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35% of normal accumulation) (Sanchez et al. 1996) (Fig. 7 B). Thus, the Shiverer mice provided a suitable model to establish whether or not RT97 phosphoepitope expression may be selectively involved in the glial signaling process that regulates regional neurofilament accumulation. Relative levels of the SMI34, SMI31, and RT97 phosphoepitopes were quantitated in NFH subunits of axonal neurofilament fractions from optic nerves of Shiverer mice lacking the MBP gene (shi/shi) (Shine et al. 1992) and mice expressing the normal level of MBP protein (Fig. 7 C). Neurofilaments from shi/shi optic axons contained 32% of the RT97 immunoreactivity detected in heterozygous or wild-type mice. This reduction in RT97 phosphoepitope levels was equivalent to the proportion of axons that fail to achieve regional neurofilament accumulation. By contrast, SMI31 and SMI34 phosphoepitope levels in NFH subunits from shi/shi mice were normal (Fig. 7 C). Immunoelectron microscopy indicated that RT97 immunoreactivity was not uniformly reduced in Shiverer axons. Axons invested by oligodendroglial cells or primitive myelin sheaths exhibited near normal levels of RT97 immunoreactivity (Fig. 7 D), whereas the 65% of axons lacking myelin or glial investment were completely unlabeled.
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Role of Subunit Stoichiometry during Regional Neurofilament Accumulation and Caliber Expansion
We found that as the proportions of NFH in neurofilaments increased during early postnatal development NFM subunit content of neurofilaments decreased commensurately, suggesting that NFH may compete with NFM in binding to NFL or forming 10-nm filaments as proposed previously by Marszalek et al. 1996. These striking changes in neurofilament composition, however, were complete nearly 2 wk before axon calibers expanded regionally. Targeted disruption of the NFH gene diminishes the radial growth of large motor axons to varying degrees (Elder et al. 1998b; Rao et al. 1998; Zhu et al. 1998), although these effects are unexpectedly small given the role suspected for NFH in radial growth. We found comparably small effects on optic axon calibers and showed that regional neurofilament accumulation was also minimally affected. However, the ability of other proteins to assume the functions of a deleted protein is not uncommon in gene ablation studies. In the case of NFH deletion, caliber reductions appear to have been offset by several compensatory mechanisms in different fiber systems. Microtubules were substantially increased (Rao et al. 1998; Zhu et al. 1998) and, in one mouse model where microtubule increases could not be demonstrated, caliber reductions were greater (Elder et al. 1998b). The loss of NFH was also accompanied by elevated NFM levels (Rao et al. 1998; Zhu et al. 1998) and possibly by greater NFM phosphorylation (Zhu et al. 1998), although, in two studies, phosphate addition to known sites on NFM (e.g., SMI31) was not increased (Elder et al. 1998b; Rao et al. 1998). The NFM compensatory response is significant in light of evidence that NFM is required to achieve normal axon caliber (Elder et al. 1998a). What is now clear from our studies of optic axons from NFH-deleted mice is that preservation of the capacity to accumulate neurofilaments regionally is accompanied by the selective appearance of the RT97 phosphoepitope on NFM, which is normally an NFH-specific phosphorylation event. Moreover, the normal 2:1 stoichiometry of NFM to NFH (Nixon and Lewis 1986) and the increase in NFM in some fiber tracts (Rao et al. 1998; our findings) results in a greater capacity of NFM to rescue NFH function in NFH-deleted mice. Even though levels of RT97 signal on NFM are lower than those on NFH, our evidence indicates that only a small proportion of the total RT97 epitope level is normally present during the period of robust neurofilament accumulation. Levels of RT97 are undetectable in Shiverer axons that are not invested with an oligodendroglial cell, whereas the smaller population of axons with primitive myelin sheaths had normal or near normal levels of RT97. These findings, therefore, suggest that axons are able to compensate partially for NFH's functions and highlight a particularly critical role of NFM and phosphorylation generating the RT97 epitope.
Delayed Appearance of the RT97 Phosphoepitope on NFH Is Selectively Associated with Regional Neurofilament Accumulation
Two distinct NFH phosphorylation events preceded appearance of the RT97 phosphoepitope and were shown to be regulated independently of both RT97-related phosphorylation and regional neurofilament accumulation. The appearance of the SMI34 phosphoepitope coincided with the first detection of NFH protein and reflected phosphorylation of KSPXX motifs along its COOH terminus (Pant and Veeranna 1995). We observed that NFH subunits initially appearing in the optic nerve had nearly peak levels of this phosphoepitope (i.e., nearly maximal SMI34/SMI33 ratios), indicating that phosphorylation at this site is essentially complete by the time transported neurofilaments reach the level of the optic nerve. This conclusion is consistent with immunocytochemical evidence that SMI34 and Erk1/2, the kinase(s) believed to generate the SMI34 epitope on NFH (Roder and Ingram 1991; Pant and Veeranna 1995; Veeranna et al. 1998), are enriched in ganglion cell perikarya (Sanchez, I., L. Hassinger, T. Wheelock, G. Hauser, and R.A. Nixon. 1996. Soc. Neurosci. 22:775.7 [Abstr.]). The appearance of SMI34 on NFH subunits still within ganglion cell perikarya suggests a role for this phosphorylation event early after NFH synthesis.
The subsequent appearance of the SMI31 epitope also preceded regional neurofilament accumulation. Our findings support the hypothesis that SMI31 generation may be related to changes in interneurofilament spacing, in agreement with earlier findings that both interneurofilament spacing and SMI31 immunoreactivity are lower at nodes of Ranvier than along internodal regions (Mata et al. 1992). Both SMI31 immunoreactivity and interneurofilament spacing were normal in Shiverer mutant mice, even though RT97 levels and regional neurofilament accumulation were markedly reduced (see Fig. 7). These observations establish that neither interneurofilament spacing nor phosphorylation represented by the SMI31 epitope regulates regional caliber expansion.
The delayed appearance of RT97 during postnatal development is the only change in neurofilament properties that coincides with the late onset of regional axon caliber expansion during development, parallels the rate of recruitment of axons for radial growth, and segregates selectively within regions of the axon that undergo neurofilament accumulation. Moreover, among the various properties of neurofilaments that change during development, only RT97 levels and regional accumulation are affected when glia-to-axon signaling is blocked in Shiverer mutant mice. When the NFH gene is deleted in mice, the RT97 epitope increases specifically on NFM, allowing this subunit to substitute partially the phosphorylation function of NFH. This result implies that additional tests of the role of KSP phosphorylation on neurofilament function using an in vivo genetic approach will require replacing both NFH and NFM with the appropriate subunits mutagenized at the appropriate sites.
The foregoing results strongly support the conclusion that phosphorylation of a subset of KSP sites is a key aspect of the regulation of neurofilament number in axons during axonal radial growth. RT97 appearance can be viewed as the culmination of a series of developmental events such as NFH expression and proper neurofilament subunit composition and SMI34/SMI31 phosphorylation, which may serve as critical antecedents to the process of neurofilament accumulation and radial growth under normal conditions. Two hypothetical functions of RT97 generation that remain to be tested are its role as an actual trigger of neurofilament accumulation or, alternatively, as a critical neurofilament modification required to stabilize the neurofilament/cytoskeletal after neurofilaments accumulate. Supporting the former possibility are observations that NFH and NFM phosphorylation slows neurofilament transport (Lewis and Nixon 1988; Archer et al. 1994). Moreover, neurofilaments containing the most highly phosphorylated isoforms of NFH, shown in this study to have the RT97 phosphoepitope, have the slowest net movement (Yabe et al. 2000) and are retained along axons for long periods (Lewis and Nixon 1988), presumably associated with a nonuniform stationary, but dynamically turning over axonal cytoskeletal network (Hirokawa et al. 1984; Nixon and Logvinenko 1986; Nixon 1998a). As proposed previously (Sanchez et al. 1996), abrupt shifts in the number of neurofilaments within a given region of the axon could be induced by local changes in a particular protein kinase or phosphatase activity that modulates the strength of interactions between neurofilaments and the axonal transport machinery, stationary axonal elements, or both. Phosphorylation may also promote stability of these neurofilaments by reducing their susceptibility to proteases (Goldstein et al. 1987; Pant 1988). The presence of the RT97 phosphoepitope on tau (Brion et al. 1993) and microtubule-associated protein 1B (Johnstone et al. 1997) during early development suggests that other potentially interacting cytoskeletal elements regulated by RT97-generating protein kinase(s) may also contribute to regional maturation of the axonal cytoskeleton. Characterizing the intercellular and intraneuronal mechanisms regulating this phosphorylation event should help to reveal how different patterns of pathologic neurofilament accumulation arise in human neurodegenerative diseases.
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These studies were supported by grant AG05604 from the National Institute on Aging, National Institutes of Health.
Submitted: 1 May 2000
Revised: 14 September 2000
Accepted: 15 September 2000
R.A. Nixon's present address is Nathan Kline Institute for Psychiatric Research, New York University School of Medicine, 140 Old Orangeburg Rd., Orangeburg, NY 10962. Tel.: (845) 398-5423. Fax: (845) 398-5422. E-mail: nixon{at}nki.rfmh.edu
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) and 700-µm (•) levels of the optic nerve in 2–3 mice were determined at different postnatal ages in 300–555 axons of calibers representative of the total optic axon population. Interanimal variation <5%. Values are displayed as total neurofilament number per 1,000 axons at each age. (G) To emphasize the regional accumulation of neurofilaments along the axons, the percentage increase in neurofilament number at the 700-µm level over the number at the 50-µm level (i.e., [700 µm – 50 µm/50 µm] x 100) for each postnatal age is plotted.
) and 700-µm (•) levels of the optic nerve (see Materials and Methods). Mean interfilament distances (SEM) are given for 1,175–2,500 neurofilaments at each axon level and postnatal age. Significant regional differences in interneurofilament spacing were detected as early as P9 (q = 2.06; Kruskal and Wallis analysis). A gradual shift to larger interneurofilament spacing was observed only at 700 µm between P9 and P16; thereafter, spacing remained unchanged (q = 2.62 and q < 0, respectively; Kruskal and Wallis analysis).
, shi) is similar to that of wild-type mice (
, wt). (B) Regional neurofilament accumulation (neurofilament number [700 µm – 50 µm/50 µm] x 100) was determined in >300 optic axons at the 50- and 700-µm levels for three wild-type or Shiverer homozygous mice. (C) SMI34, SMI31, and RT97 immunoreactivities as a ratio of total NFH (SMI33) immunoreactivity were determined in the same groups of mice by quantitative densitometry. All values are expressed relative to the wild-type control set at 100%. Each value represents the mean and standard error from four separate samples (*P < 0.05 and #P > 0.05 by Student's t test). Immunoelectron microscopy of the RT97 epitope in axons of normal and homozygous Shiverer mice. After incubation of treated epon sections with RT97 (see Materials and Methods), 5-nm gold-conjugated secondary antibody decorated neurofilaments in normal optic axons (not shown) and (D) Shiverer axons associated with primitive myelin sheaths (my; arrowhead). No RT97 immunoreactivity was detected in unmyelinated axons (unmy) from Shiverer mice.