Caspr and Contactin Are Distinctly Expressed by Neurons and Myelinating Glia
We first determined the expression patterns of contactin/ F3/F11 (contactin) and Caspr using well-characterized antibodies that recognize the extracellular and the cytoplasmic domains of these proteins, respectively. Purified cultures of sensory neurons, Schwann cells, oligodendrocyte progenitors, and differentiated progenitors (consisting of both O4+/O1− prooligodendrocytes and O1+ oligodendrocytes) were prepared, and expression was analyzed by immunofluorescence (Fig. ). Contactin is expressed robustly by neurons (Fig. A), by oligodendrocytes (Fig. E), and by their progenitors but not by Schwann cells (Fig. C). By contrast, Caspr expression is restricted to neurons and their processes (Fig. B); no staining of Schwann cells (Fig. D) or of cells in the oligodendrocyte lineage (Fig. F) was observed. Both Caspr and contactin are diffusely distributed along the entire surface of neurons and their processes.
Figure 1 Expression of contactin and Caspr by neurons, Schwann cells and oligodendrocytes. Primary cultures of sensory neurons (A and B), Schwann cells (C and D), and oligodendrocytes (E and F) were stained with antibodies to contactin (A, C, and E) and Caspr (more ...)
Myelination Regulates the Expression and Distribution of Caspr
We next examined contactin and Caspr expression in established cocultures of Schwann cells and DRG neurons (Fig. ). These cultures contained significant numbers of myelinated fibers as visualized by staining for myelin basic protein (MBP), which is a component of compact myelin (Fig. , B and D). Contactin (Fig. A) is significantly downregulated in the cocultures in both ensheathed and myelinated fibers compared with isolated primary neurons. Contactin continues to be expressed at relatively high levels on the occasional unensheathed neurite that persists in such cultures (Fig. A, asterisks). These results indicate that direct Schwann cell contact is responsible for the downregulation of contactin expression.
Figure 2 Contactin and Caspr expression in myelinating cocultures. Schwann cells were added to cultures of dissociated sensory neurons and allowed to repopulate the neurites. Ascorbic acid and serum were added to promote myelination, and cultures were fixed (more ...)
Caspr expression is also significantly reduced in the cocultures (Fig. C). Of particular note, there is a dramatic change in its distribution, with very high levels present at the ends of the myelin sheaths. This concentration of Caspr is located just beyond the compact myelin sheath (as visualized by MBP staining) in the region of the paranodes. In the case of isolated myelin segments, Caspr is present at either end of the myelin segment (Fig. , C and D, arrowheads). Where two myelin segments approach each other to form a node, Caspr staining appears as a doublet. Two representative nodes are indicated by the arrows in Fig. , C and D, and a higher power view of the node in the center of the field is shown in the inset. Of note, the Caspr staining is found within the gap of MBP staining but does not appear to extend into the node itself.
Caspr Is Downregulated with Myelination and Is Associated with a Detergent Insoluble Fraction
To assess more accurately the relative levels of expression of these proteins in the cultures, we performed Western blotting using 125I-labeled protein A as a reporter (Fig. ). We compared culture lysates (50 μg) of neurons, Schwann cells, and Schwann cell/neuron cocultures after 1 or 3 wk in myelinating conditions; lysates (25 μg) of oligodendrocyte progenitors and differentiated O4+ oligodendroglia were also analyzed (Fig. A). Caspr migrated with an expected molecular mass of ~190 kD (Fig. A, upper panels); in several experiments we also observed a minor band of ~50 kD that may be a proteolytic fragment (data not shown). Consistent with immunostaining results, DRG neurons expressed Caspr at robust levels, whereas it was undetectable in Schwann cell and oligodendrocyte lysates. Caspr expression was greatly reduced in the cocultures compared with the neurons (more than 10-fold in several experiments). A progressive reduction in Caspr levels was also noticeable between 1 and 3 wk of coculture.
Figure 3 Immunoblot and Northern analysis of contactin and Caspr expression. (A) Caspr and contactin expression in cultured cells and association of Caspr with the detergent insoluble complex. 50 μg of protein lysate prepared from neuron (DRG), Schwann (more ...)
In parallel, we examined whether Caspr might be part of a cytoskeleton-enriched, detergent-insoluble complex. We extracted neuron cultures and myelinating cocultures (at 4 wk) with 1% Triton X-100 (Fig. A, lanes T), solubilized the remaining material with SDS (Fig. A, lanes S), and analyzed both fractions by Western blotting (Fig. A, top right). Only ~25% of the total Caspr in the neuron cultures was extracted by Triton (Fig. A, compare lane DRG:T to DRG:S). In the myelinating cocultures, less than 10% was extracted by Triton ( Fig. A, compare lane 3 wk:T to 3 wk:S), suggesting that even more of the Caspr is part of a detergent-insoluble complex. In related studies, we have found that Caspr staining persists in the cocultures after Triton extraction, whereas MBP and MAG are completely removed (data not shown). Likewise, extraction of brain membrane fractions with a variety of nonionic detergents (e.g., Triton, Brij, digitonin, octylglucoside) released less than half of the total Caspr in each case (data not shown). These results indicate that Caspr is associated with a detergent-insoluble, cytoskeleton fraction in vitro and in vivo.
We also examined contactin expression by Western blotting. Contactin is expressed by neurons and oligodendrocytes, but not by Schwann cells, and is similarly progressively downregulated in the cocultures (Fig. A
). Of note, contactin is detected as a doublet on neurons and as a single band on oligodendrocytes. We had previously found that the upper band of this doublet is removed by phosphatidylinositol phospholipase C, whereas the lower band is phosphatidylinositol phospholipase C–resistant and is likely a preform of contactin (Rosen et al., 1992
). As shown in Fig. B
, Caspr and contactin expression also significantly decrease during postnatal sciatic nerve development (day 1 through adulthood). At all times, they are present at substantially reduced levels in sciatic nerves when compared with hippocampus (Fig. B
, lane Hc
) used as a CNS control.
A Northern blot for contactin (Fig. C
) confirms its high level expression in oligodendroglia; a major band of ~6.4 kb and minor bands between 3–4 kb were present, consistent with previous reports (Gennarini et al., 1989
). No expression of contactin was detected in Schwann cells or at varying times of sciatic nerve development. By contrast, Caspr mRNA of the expected size (6.2 kb) was detected in the CNS control but not in glia or sciatic nerve samples (data not shown). These results are consistent with the protein studies described above and indicate that Caspr and contactin in sciatic nerve are exclusively of axonal origin.
Redistribution of Caspr during Myelination
To investigate further the mechanisms by which Caspr becomes concentrated at the paranodes, we have analyzed its distribution at different times of myelination in the coculture system (Fig. ). Schwann cells were seeded onto preestablished networks of DRG neurites and maintained for several days in a defined media in which Schwann cells proliferate but do not ensheathe or myelinate nerve fibers. Ascorbic acid was then added (day 0) to initiate ensheathment and myelination. Cultures were fixed at various times thereafter and double stained for Caspr and MAG, a myelin-specific protein that is expressed at the onset of myelination (Owens and Bunge, 1989
Figure 4 Redistribution of Caspr during myelination in vitro. Schwann cells were added to cultures of dissociated sensory neurons and allowed to repopulate the neurites. Ascorbic acid and serum were added to promote myelination, and cultures were fixed after (more ...)
New myelin sheaths begin to form on days 3 to 4 in the cocultures and can be detected by their expression of MAG (Fig. B, asterisks). Caspr is initially diffusely expressed along the entire neurite at this time, although at somewhat reduced levels compared with pure populations of neurons (Fig. A). Interestingly, in some instances there appeared to be a slight increase in Caspr expression under some of the forming segments of myelin (data not shown). By 6–7 d, myelin sheaths have begun to compact, as indicated by the redistribution of MAG into the periaxonal glial membrane, Schmidt-Lanterman incisures and the paranodal loops. At the same time, Caspr began to accumulate into the paranodes of a few myelinated segments. (Examples of nascent paranodes are indicated by the arrowheads in Fig. C.) Of additional note, Caspr expression frequently appeared attenuated at the center of corresponding internodes.
By 11 d, there is a striking accumulation of Caspr into multiple paranodes (Fig. , E and F). Concentrations of Caspr were invariably associated with well-myelinated segments, particularly those associated with MAG-positive Schmidt-Lanterman incisures, and accumulated into both paranodes of a myelinated segment at approximately the same time. Expression in the internode was reduced in those segments containing concentrations of Caspr at the paranodes compared with other segments that were still in the process of myelination. Two examples are indicated by arrows in Fig. , E and F, which mark paranodal accumulations. It may be seen that the mature myelin internodes located above the arrows express less Caspr than the two nascent myelin internodes located below the arrows. Taken together, these results indicate that Caspr accumulation in the paranodal region is a late event that occurs with myelin compaction and maturation and is likely to reflect a redistribution from the internode.
Caspr Is Concentrated in Paranodes in the CNS and PNS
To determine whether Caspr is localized at the paranodes of myelinated fibers in the PNS and CNS in vivo, whole mounts of teased rat sciatic nerve fibers and sections through various regions of the rat brain were immunolabeled with anti-Caspr antibody and examined by light microscopy. Intense paranodal staining for Caspr was observed in sciatic nerve fibers (Fig. A), and virtually all of the fiber tracts in the brain, including the large myelinated axons of the facial nerve (Fig. B) and the small, thinly myelinated axons of the corpus callosum (Fig. A). Minimal staining, if any at all, was evident in the internodes. Paranodal staining was not observed with the corresponding control preimmune serum (data not shown). Although the protein appeared to be absent from the outer surfaces of the paranodal loops, it could not be determined at the light microscopic level whether Caspr immunoreactivity was associated with the inner surfaces of the paranodal loops adjacent to the axon, the axonal membrane, or with both. More precise localization of the protein was resolved by immunoelectron microscopy (see below).
Figure 5 Caspr is concentrated in the paranodal regions of myelinated fibers in the PNS and CNS. A teased fiber preparation of adult sciatic nerve (A) and a coronal section through the facial nerve in the pons (B) were stained with an antiserum against Caspr (more ...)
Figure 6 Contactin/F3 is not concentrated in the paranodal region. Staining of the corpus callosum (cc) with antisera against Caspr and contactin/F3 is shown. Caspr staining (A) is concentrated in the paranodal regions of the small myelinated fibers of the corpus (more ...)
In addition to staining of the fiber tracts, low to moderate levels of diffuse Caspr immunoreactivity were present throughout the neuropil of the brain. An example of such diffuse staining in the lateral septal nucleus is shown in Fig. A (SN). Considerable punctate staining, most likely representing paranodal regions of small myelinated fibers but possibly representing other neuronal structures, was found throughout the neuropil. In general, neuronal cell bodies in most regions of the brain examined were either unlabeled or only lightly labeled by the Caspr antibody.
The distribution of contactin immunoreactivity in brain sections was also examined by light microscopy (Fig. B). In contrast to Caspr, and consistent with the in vitro studies described above, contactin was not concentrated at paranodal regions of myelinated fibers in these sections. However, relatively strong contactin staining of cells, possibly interfascicular oligodendrocytes, was observed in fiber tracts such as the corpus callosum (Fig. B inset, arrows). Light to moderate contactin immunostaining was also prominent in many neurons distributed throughout the brain, such as those of the cerebral cortex (Fig. B).
Caspr Is Localized to the Septate-like Junctions of the Paranodes
To determine more precisely the localization of Caspr within the paranode and to distinguish whether it was associated with neuronal and/or glial cell membranes, the distribution of the protein in the corpus callosum and the facial nerve (within the pons) was examined by immunoelectron microscopy. The results of these studies confirmed the light microscopic observations that Caspr is primarily concentrated at the paranodal regions of myelinated fibers. Furthermore, they showed that the protein is a component of the axonal membrane and not that of the paranodal loops of the glial cell. Representative nodal regions of myelinated fibers in the corpus callosum showing the silver-enhanced immunogold particles denoting the distribution of Caspr are shown in Fig. , A–C. Similar patterns of paranodal labeling were observed in sections of the facial nerve (data not shown). Most of the immunolabeling at nodal regions was restricted to the inner surface of the axonal membrane containing the septate-like junctions. An example of the characteristic septae of these junctions are shown at high magnification in the inset of Fig. A. This staining of the inner membrane surface is expected given the reactivity of the antibody with cytoplasmic determinants on Caspr. In favorable planes of section, such as that shown in Fig. B, strikingly intense immunolabeling was observed along the presumptive inner surface of the paranodal axonal membrane. Importantly, virtually no labeling was observed in the nodal gap itself and only occasional particles were detected in the axonal membrane or cytoplasm in the internodes of myelinated fibers. Taken together, these results indicate that Caspr is likely to be a component of the septate-like junctions at the paranodal region.
Figure 7 Immunoelectron microscopic localization of Caspr in myelinated and unmyelinated nerve fibers of the corpus callosum. Representative longitudinal sections of nodal regions of myelinated fibers with silver enhanced immunogold particles denoting the (more ...)
In addition to its concentration at the paranodal region of myelinated fibers, rather intense Caspr immunogold labeling was also observed around the inner surfaces of axonal membranes of some small-diameter nerve fibers that appeared to be unmyelinated. Examples of such fibers from the corpus callosum cut in cross section are shown in Fig. D
). These labeled profiles are unlikely to represent cross sections through the paranodal regions of myelinated fibers because they appear to lack the increased number of microtubules in the axon or surrounding myelin lamellae characteristic of this region (Peters et al., 1991
). Furthermore, these profiles were often present in clusters within the callosum and much more frequent than would be expected for paranodes, which are quite short in comparison to the length of nerve fibers. These findings suggest that unmyelinated or ensheathed axons in the CNS may continue to express considerable amounts of Caspr and are consistent with the in vitro studies above, which showed that neurons grown in the absence of Schwann cells or those during the early stages of myelination express significant levels of this protein.