BACKGROUND
nervous system function depends on proper molecular organization between neurons and glial cells.
in myelinated neurons, the segregation and enrichment of proteins in the defined domains, the node of ranvier, paranode, and juxtaparanode, is critical for saltatory action potential propagation
<cit> .
without proper organization, electrical nerve conduction is delayed and can result in significant motor and sensory deficits.
the paranodal domain is a region of direct interaction between the glial myelin membrane loops and neuronal plasma membrane.
axo-glial septate junctions  link these glial membrane loops to the axonal membrane and establish the paranodal ionic barrier separating sodium  channel-enriched node from the potassium  channel-enriched juxtaparanode
<cit> .
agsj’s are composed of three transmembrane proteins: contactin
<cit> , contactin-associated protein
<cit> , and neurofascin <dig>
<cit> .
genetic ablation of any of the three molecules disrupts the paranodal barrier function and results in degraded action potential propagation
<cit> .
while much research has focused on these three proteins, other molecules contribute to domain formation and stabilization.
for example, caspr <dig> and tag- <dig> are required for the organization of the juxtaparanodal domain and localization of potassium  channels
<cit> .
for long-term stability, caspr and caspr <dig> are thought to rely on cytoskeletal scaffold proteins to link their associated complexes with the axonal cytoskeleton.
loss of caspr results in significant cytoskeletal disorder
<cit> , and since its extracellular partner cont lacks an intracellular c-terminus, the c-terminus of caspr is likely a critical region of interaction between agsj’s and the axonal cytoskeleton.
previous studies using  <dig> b mutant mice revealed that the  <dig> b cytoskeletal protein contributes to paranodal and juxtaparanodal stability
<cit> .
a recent report also highlights  <dig> g as an organizer of internodes in the peripheral nervous system
<cit> .
the neuron is abundant with cytoskeletal scaffolds however, and such findings do not exclude the possibility that other cytoskeletal elements may contribute to paranodal maintenance and long-term stability.
whrn is a cytoskeletal scaffold protein that plays an important role in vision and hearing
<cit> .
in humans, whirlin   mutations have been linked to usher syndrome type ii , an autosomal recessive vision-hearing impairment disorder
<cit> .
in mice, the whrn coding sequence consists of  <dig> exons with two dominant splice variants, a full length and short  isoform.
both variants contain a proline-rich domain and pdz-domain which have been shown to link submembranous cytoskeletal elements to transmembrane complexes, as well as to self-oligomerize
<cit> .
in the eye, whrn interacts with the transmembrane proteins usherin  and very large g protein-coupled receptor- <dig>  within the periciliary membrane complex of photoreceptors.
likewise in the inner ear, whrn interacts with usherin and vlgr1/gpr <dig>  which form the stereociliary ankle-links.
additionally, whrn interacts indirectly with  <dig> b
<cit>  and  <dig> r through mpp1/p55
<cit> , and directly with myosin xva
<cit> .
finally, whrn is implicated with esp <dig> in stereocilia-length regulation
<cit> .
while the function of whrn in the ear and the eye has received significant attention, very little is known about its function in the central and/or peripheral nervous system.
there are reports of whrn protein expression in the cerebrum, cerebellum, and brainstem in wild-type mice and the protein is absent in whrn knockout  and whirler  mutant mice
<cit> .
in drosophila, the closest homolog to whrn is dyschronic
<cit> .
in dysc mutants there is arrhythmic locomotor behavior but the eclosion circadian rhythms and clock protein cycling is unaffected
<cit> .
here we report that whrn is involved in proper compaction of the paranodal region in myelinated axons and for proper stabilization of the axonal cytoskeleton.
RESULTS
whrn is expressed in central and peripheral nervous system tissues throughout development
whrn is a cytoskeletal scaffolding protein which functions to link membrane protein complexes to the cytoskeleton within hair cell stereocilia of the ear and photoreceptors in the eye.
to begin assessing its function in myelinated neurons, we obtained whrn exon  <dig> knockout mice
<cit> .
as reported previously, the murine whrn locus consists of  <dig> exons with two dominant splice variants, a full length  isoform and a short  isoform .
both variants contain pdz-domains  and a proline-rich domain .
after initial back crossing to c57bl <dig> mouse strain, we identified and confirmed the whrn genotype using pcr methods.
to begin characterizing mrna expression of whrn in the central  and peripheral nervous systems , we examined the relative expression of whrn by reverse transcriptase polymerase chain reaction  in dorsal root ganglia , sciatic nerves , and spinal cord  tissues  in postnatal 21 day-old mice.
with this subset of tissues we could delineate the origin of whrn expression as sc tissue is a combination of glial and neuronal nuclei, drg is predominantly neuronal, and sn is principally glial cytoplasm and nuclei.
since whrn has two major isoforms, we designed specific primers to distinguish the full length isoform  alone and those common to the full length and short isoforms .
no expression of whrn isoforms was observed in whrn−/− mice .
robust expression of whrn  mrna was observed in drg and sc tissue while weak expression was observed in sn tissue.
interestingly, weak expression of whrn  mrna was observed in drg and sc tissue but no significant expression was observed in sn tissue.
actin  mrna expression was used as a control for total rna present.
relative expression was quantified as a ratio of whrn to actin mrna between three total rt-pcr analyses .
after finding whrn mrna expression in the wild-type cns and pns neuronal tissues, we next sought to determine its protein expression in p <dig> whrn+/+ versus whrn−/− mice by immunoblot analysis.
in order to pursue these experiments, we generated several antibodies and affinity-purified one to the non-domain encoding, c-terminal region  of whrn .
after affinity-purification, wild-type  and whrn knockout  lysates derived from drg, sn, sc, and whole eye were immunoblotted .
whole eye lysates were used as a positive control for protein expression based on previous reports
<cit> .
a 110 kda whrn band representing the full length protein was present in drg, sc, and whole eye lysates.
no 110 kda whrn band was observed in whrn−/− lysates or in wild-type sn lysate.
each tissue type showed similar total protein levels between genotypes based on total tubulin on immunoblots .
having confirmed whrn−/− tissues were deficient in whrn mrna and protein, we next sought to determine if loss of whrn resulted in altered protein expression of known nodal, paranodal, and juxtaparanodal proteins.
we prepared spinal cord lysates from  <dig>   <dig>   <dig>  and 16 week-old wild-type  and whrn knockout  mice and immunoblotted for whrn  .
whrn protein expression levels were similar from  <dig> to 16 weeks in wild-type mice while no expression was observed in the whrn−/− at any time point.
next, we immunoblotted for the paranodal protein caspr  and found similar expression levels between 4–16 week-old in both whrn+/+ and whrn−/− mice.
using a pan-neurofascin-c-terminal  antibody, we observed similar levels of nodal neurofascin <dig> and paranodal neurofascin <dig> protein expression in wild-type and whrn knockout mice across 4–16 weeks of age.
examination of neuronal cytoskeletal protein  <dig> b and juxtaparanodal protein caspr <dig> shows steady protein expression levels in wild-type and whrn knockout mice from 4–16 weeks of age.
finally, previous reports showed in vivo interaction between rat whrn/cip <dig> and calmodulin-dependent serine kinase
<cit> , a synaptic organization protein.
after immunoblotting for cask, no difference in its protein level was observed across whrn genotype or ages from 4–16 weeks.
total protein levels in spinal cord lysates were consistent across the ages and genotypes based on tubulin levels .
in summary, these developmental expression profiles suggest that loss of whrn expression does not affect the overall expression or stability of other axonal domain markers.
1b expressed as a ratio of whrn to actin band intensity.
d. immunoblots of 110 kda whrn protein band in wild-type and whrn mutant drg, sn, sc, and whole eye using affinity-purified rbwhrn <dig> antibody.
αtubulin served as a loading control.
e. immunoblots of wild-type and whrn knockout mutant  <dig>   <dig>   <dig>  and 16-week-old spinal cord lysates.
the expression profile using various myelinated axonal domain markers includes caspr, neurofascin ,  <dig> b, caspr <dig>  as well as cask.
αtubulin served as a loading control.
f. immunofluorescence of teased sciatic nerve fibers from wild-type  and whrn knockout mice  mice.
neurofascin  and paranodal caspr  reveal paranodal compaction is disrupted in whrn knockout fibers.
note nfct  detects both paranodal nf <dig> and nodal nf <dig> isoforms.
whrn knockout mice reveal a quantifiable paranodal compaction phenotype in peripheral myelinated axons
to determine the effects of whrn loss on axonal domain organization, we examined well-characterized nodal, paranodal, and juxtaparanodal markers by immunofluorescence in wild-type and whrn−/− mice.
extensive and repeated immunostaining with our whrn antibody revealed no consistent localization or differences in whrn localization between wild-type to whrn−/− fibers .
compared to wild-type, the most striking observation in teased whrn−/− sciatic nerve fibers was the spring-like separation  of the paranodal axo-glial septate junction  loops beginning along the paranodal-juxtaparanodal border as observed by caspr and nf <dig> immunostaining.
irregular paranodal compaction is rarely observed in wild-type fibers, so we sought to quantify the overall observation of this phenotype in whrn+/+ and whrn−/− fibers.
we utilized a blinded counting strategy to count spring-like phenotypes from wild-type  or whrn−/−  caspr-stained paranodes.
a statistically significant difference  was observed in the percentage of irregular paranodal compaction at  <dig> %  in wild-type fibers compared to  <dig> %  in whrn knockout mice.
having confirmed the significance of this spring-like paranodal phenotype in 7 week-old whrn knockout fibers, we expanded our analysis to 4–8 week-old time points.
sciatic nerves from whrn+/+ or whrn−/− littermate mice at  <dig>   <dig>  and 8 weeks, were immunostained with antibodies against nodal, paranodal, and juxtaparanodal markers .
in the 4 week-old wild-type sciatic nerve , juxtaparanodal kv <dig>   was separated from nodal nf <dig>  by paranodal caspr .
this demarcation of domains  in myelinated fibers is also observed in 4 week-old whrn−/− fibers .
myelin loops of agsjs in 4 week-old whrn−/− sciatic nerve fibers  were already separating away from the paranode compared to 4 week-old wild-type sciatic nerve fibers .
breaks in kv <dig>  localization  were observed even where caspr staining  was limited to a single myelin loop, suggesting agsj’s barrier function was conserved.
juxtaparanodal localization was diffuse and asymmetric in whrn−/− fibers compared to wild-type .
when comparing wild-type to whrn knockout mice, we observed no obvious differences in intensity levels of juxtaparanodal  or nodal  markers.
at 6 weeks, teased wild-type sciatic nerves  showed typical domain organization.
6 week-old whrn null sciatic nerves have typical nf <dig>  nodal domain organization while caspr paranodal compaction  and kv <dig>  juxtaparanodal  localization were disrupted.
additional immunostaining in 6 week-old whrn null sciatic nerves with a pan-neurofascin-c-terminal  antibody  detected both nodal nf <dig> and paranodal nf <dig>  paranodal nf <dig>  and caspr  immunostaining mostly colocalize  in whrn−/− sciatic nerve fibers and revealed similar problems with paranodal compaction.
ankg nodal  domain organization in 6 week-old whrn−/− sciatic nerve fibers appeared similar to wild-type .
finally, immunostained 8 week-old sciatic nerves revealed similar nf <dig> nodal  and ankg nodal organization .
like  <dig> and 6 week-old sciatic nerves, caspr  and nfct  immunostaining consistently revealed paranodal compaction defects as well as kv <dig>  juxtaparanodal  diffusion in whrn knockout mice when compared with wild-type .
to determine if such peripheral nerve phenotypes could be the result of differences in inner mesaxons, we immunostaining 7 week-old wild-type and whrn knockout fibers with mag but observed no striking phenotypic differences in localization .
in summary, the paranodal and juxtaparanodal regions displayed phenotypes that suggest that normal compaction of the paranodal loops fails to occur in whrn knockout mice at  <dig>   <dig>  and 8 weeks of age.
during initial quantification we observed that larger diameter myelinated fibers had proportionally more paranode compaction defects compared to thinner caliber myelinated fibers, so we imaged and assembled twenty caspr-stained confocal images for each genotype and time point  to assess any subtle, sub-micron paranodal changes by light microscopy due to whrn loss.
next, we measured various dimensions of the paranode .
note images collected were from ~ <dig> micron caliber myelinated neurons.
using zeiss software, micron measurements were recorded, tabulated, and reported as averages with standard deviations for the nodal gap , the paranodal diameter , paranodal width , as well as the percentage of paranodes which display paranodal compaction abnormalities .
the nodal gap  for wild-type and whrn−/− sciatic nerves was  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 4 weeks,  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 6 weeks, and  <dig> ± <dig>  μm and  <dig> ± <dig>  at 8 weeks respectively.
no statistically significant difference was noted between ages or genotypes with respect to nodal gap.
the paranodal diameter  for wild-type and whrn−/− sciatic nerves was  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 4 weeks,  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 6 weeks, and  <dig> ± <dig>  μm and  <dig> ± <dig>  at 8 weeks respectively.
with respect to paranodal diameter, measurements were similar between all groups.
the paranodal width  for wild-type and whrn−/− sciatic nerves was  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 4 weeks,  <dig> ± <dig>  μm and  <dig> ± <dig>  μm at 6 weeks, and  <dig> ± <dig>  μm and  <dig> ± <dig>  at 8 weeks respectively.
no statistically significant difference was found in paranodal width.
the paranodal compaction phenotype percentage  for wild-type and whrn−/− sciatic nerves was 15%  and 55%  at 4 weeks, 15%  and 50%  at 6 weeks, and 15%  and 55%  respectively.
as a reminder, the increase in the whrn knockout phenotype percentage represents the shift in selection from all fibers  to larger-caliber myelinated fibers .
note the greater percentage of paranodes with compaction issues in whrn−/− sciatic nerves  likely contributes to the increased deviation in paranodal widths .
additionally, comparisons between 8 week-old wild-type and whrn−/− sciatic nerve fibers demonstrated no significant difference in the conduction velocity  or waveforms  of measured compound action potentials in two separate measurement trials.
whrn knockout mouse sciatic nerve and spinal cord myelinated fibers display paranodal compaction abnormalities throughout development
to expand on the characterization of whrn loss with respect to myelinated domain organization, we examined a larger developmental window from postnatal ages 2 weeks to 1 year.
wild-type and whrn knockout sciatic nerves revealed the following percentages of caspr-stained phenotype-positive paranodes :  <dig> % and  <dig> % at 10 weeks,  <dig> % and  <dig> % at 20 weeks,  <dig> % and  <dig> % at 30 weeks,  <dig> % and  <dig> % at 40 weeks, and  <dig> % and  <dig> % at 1 year, respectively.
similar to the 4–8 week studies , we immunostained fibers with nodal, paranodal, and juxtaparanodal markers, as well as the axonal cytoskeletal protein markers  <dig> b and heavy chain neurofilament  given whrn’s known cytoskeletal scaffolding role.
we observed no differences in nodal formation using nf <dig>  in 2 week-old fibers.
abnormalities in paranodal formation and com-paction were observed in caspr-stained whrn−/− fibers  compared to wild-type .
overall kv <dig>  juxtaparanodal signal appeared similar between 2 week-old wild-type  and whrn−/− fibers  with accumulation and enrichment of kv <dig>  channels  neighboring caspr within the internodal region.
no obvious difference in intensity or localization of nfl-h  or  <dig> b  was found in 2 week-old sciatic nerve fibers between whrn genotypes.
with 10 week-old sciatic nerve fibers, we observed no differences in nodal domains using nf <dig> .
like 8 week-old fibers , we observed 10 week-old whrn−/− fibers with abnormal paranodal compaction , when compared to wild-type fibers .
juxtaparanodal domains stained with kv <dig>   appear more diffuse but similar in overall intensity in 10 week-old whrn−/− fibers when compared to wild-type.
we observed slight enrichment of nfl-h  and  <dig> b  within the paranodal region of 10 week-old wild-type sciatic nerves compared to whrn knockout mice, but the overall intensity of nfl-h and  <dig> b along the remaining axon appeared similar.
immunostaining of  <dig>   <dig>  and 40 week-old wild-type sciatic nerve fibers were performed and domain organization was identical to 10 week-old wild-type fibers .
looking at  <dig>   <dig>  and 40 week-old whrn−/− peripheral nerve fibers, we observed no differences in nodal domains between whrn wild-types  or knockout mice using nf <dig> .
similar to 10 week-old fibers , we observed numerous  <dig>   <dig>  and 40 week-old whrn null fibers stained with caspr  with abnormal paranodal compaction when compared to wild-type fibers .
in  <dig>   <dig>  and 40-week old whrn−/− fibers, juxtaparanodal domains stained with kv <dig>   showed numerous breaks at sites of caspr signal, appeared less symmetrical, and displayed similar overall signal when compared with wild-type .
cytoskeletal marker nfl-h staining  was generally uniform within the axon, and  <dig> b  was present in all axonal domains except the node in  <dig>   <dig>  and 40 week-old whrn−/− sciatic nerves.
one-year-old sciatic nerves showed no difference in nodal organization .
one year-old whrn−/− sciatic nerves  revealed abnormal paranodal compaction compared to wild-type fibers .
notably, we observed paranodal compaction defects in older whrn−/− sciatic nerve fibers  like blocks and bulges in caspr signal as well as the spring-like phenotype observed in younger fibers .
as seen previously, the juxtaparanodal domains of whrn−/− peripheral nerves stained with kv <dig>   showed significant asymmetric enrichment and periodic breaks as well as overlap with caspr signal when compared with one year-old wild-type fibers .
also, patchy cytoskeletal-deficient staining was observed in older whrn−/− fibers  near the paranode.
cytoskeletal marker nfl-h staining was weakly enriched within the paranode of the wild-type axon  and less uniform within whrn−/− fibers with paranodal compaction problems .
likewise,  <dig> b showed disrupted juxtaparanodal cytoskeletal staining in one year-old whrn−/− fibers  which was not observed in the wild-type fibers .
we also sought to examine the effects of whrn loss on axonal domain organization in the central nervous system .
utilizing white matter tracts in the spinal cord, we were able to identify by immunostaining subtle but consistent differences in paranodal compaction.
as in the pns , we stained longitudinal spinal cord sections with nodal, paranodal, and juxtaparanodal markers .
wild-type and whrn knockout spinal cord fibers revealed the following percentages of caspr-stained phenotype-positive paranodes :  <dig> % and  <dig> % at 10 weeks,  <dig> % and  <dig> % at 20 weeks,  <dig> % and  <dig> % at 30 weeks, and  <dig> % and  <dig> % at 40 weeks.
no differences in nodal organization were observed  in 2 week-old spinal cord sections.
at  <dig>   <dig>   <dig>  40 week-old, nodal organization appeared similar between wild-type and whrn−/− spinal cord fibers using nf <dig> .
immunostaining with paranodal caspr revealed subtle and infrequent paranodal abnormalities in 2 week-old whrn null spinal cord fibers  compared to wild-type .
in contrast, obvious and regular paranodal compaction defects were observed in  <dig> ,  <dig> ,  <dig> , and 40 week-old  whrn−/− spinal cords compared to their wild-type controls.
finally, juxtaparanodal kv <dig>  immunostaining of  <dig> ,  <dig> ,  <dig> , and 40 week-old  wild-type spinal cord myelinated axons showed similar overall expression as whrn knockout axons, but consistent juxtaparanodal disorganization and colocalization with loosened caspr-stained myelin loops was observed most often in whrn−/− fibers.
in summary, there is significant evidence that loss of whrn disrupts normal paranodal compaction in 10–40 week-old myelinated spinal cord axons with subsequent effects on juxtaparanodal organization.
taken together, both the peripheral and central nervous systems likely utilize the cytoskeletal properties of whrn to help stabilize the cellular organization between myelinating glial cells and neurons around the paranodal region throughout development.
whirlin knockout mice have cerebellar purkinje cells with bead-like, axonal swellings
to determine the effects of whrn loss on cerebellar purkinje cell morphology, we immunostained cerebellar slices from 6 week-old wild-type, whrn knockout, and double whrn and  <dig> b <cit>  null animals .
given that caspr <cit>  and cgt <cit> , two genes critical for formation of a proper paranode, display purkinje axonal swellings and cytoskeletal disorganization, we were curious as to the effects of the combined loss of whrn and  <dig> b. while no obvious differences in localization was observed using our whrn antibody  in any of these genotypes, we immunostained against  <dig> b , purkinje-specific calbindin , and glial-specific myelin basic protein/mbp .
there was no difference in  <dig> b staining intensity  between wild-type and whrn knockout slices.
upon examining whrn−/− cerebellum sections , purkinje axons appeared to contain bead-like, swellings along their extensions through the granular layer using both calb  and mbp  in comparison to a uniform caliber axon in wild-type fibers .
the secondary loss of  <dig> b protein resulted in more swellings observed with calb  and mbp  when compared with whrn null animals alone.
in summary, the cytoskeletal elements whrn and  <dig> b likely have an assistive role in preventing cytoskeletal accumulation and disorganization within cerebellar purkinje cell axons in a similar phenotypic manner to caspr null mice
<cit> .
ultrastructural abnormalities in whrn knockout sciatic nerve, spinal cord fibers, and cerebellar purkinje axons
to further understand whrn’s role in myelinated axons, transmission electron microscopy was performed in order to examine the ultrastructural architecture in myelinated axons in the pns  and cns  of 7 week-old and 3 month-old wild-type and whrn−/− mice.
low-magnification, cross-section electron micrographs of wild-type  and whrn knockout  myelinated sciatic nerve fibers showed the typical organization of tightly bound, electron-dense myelin wraps around the internodal region of the axonal membrane.
accumulation of mitochondria and lipid vesicles  in the internodal regions was clearly observed in whrn knockout animals compared to wild-type sciatic nerve fibers.
given the potential role of the mesaxon in the observed light microscope phenotype
<cit> , we found no striking differences in the ultrastructural organization or arrangements of the inner mesaxon along the internodal region at 7 weeks or 3 months of age .
consistent with our immunostaining data, no obvious differences in nodal organization were observed in either genotype .
higher magnification along the wild-type paranodal region  revealed the hallmark electron-dense agsjs formed between the glial paranodal loops and axolemma and accompanying parallel arrays of cytoskeletal elements in the axon.
in contrast, the whrn knockout paranodal region of 7 week-old  and 3 month-old  displayed poorly defined but present agsjs , less organized neurofilaments and microtubules, and consistent accumulation of mitochondria and lipid vesicles  in the paranodal region.
the ultrastructural phenotypes displayed at both 7 weeks and 3 months of age suggest that whrn is important for the long-term maintenance and the overall structure of the myelinated axons.
in the central nervous system, low-magnification, electron micrographs of wild-type  and whrn knockout  myelinated fibers showed slight differences in spinal cord cross-sections.
like the peripheral nerves, the overall organization between glial cell and neuron remained similar between wild-type and whrn−/− fibers.
however, mitochondria were slightly more abundant in whrn knockout fibers.
no obvious ultrastructural differences were observed in the node of either genotype , but greater accumulation of mitochondria  and lipid vesicles was observed in the paranodal and juxtaparanodal regions.
higher magnification along the paranodal region  revealed the expected electron-dense agsjs formed between myelin loops and the axolemma.
like in the pns, the whrn knockout paranodal region of 7 week-old  and 3 month-old  displayed poorly defined but present agsjs  and accumulation of mitochondria and lipid vesicles  in the paranodal region.
examining the cerebellum, we observed purkinje axon swellings in whrn knockout fibers .
low-magnification electron micrographs of 7 week-old wild-type  and whrn knockout at 7 weeks  and 3 months  of age showed striking differences in purkinje axon myelinated fibers in the cerebellum granular layer.
this region shows axonal swellings filled with densely-packed organelles, particularly mitochondria and vesicles .
taken together, the ultrastructural analyses of whrn knockout mice demonstrate that whrn is critical for the stability of paranodal organization, proper axonal cytoskeletal arrangements, and prevention of sub-cellular organelle accumulation in myelinated axons.
CONCLUSIONS
cellular and molecular interactions between neurons and glia establish and stratify the numerous tasks of the nervous system.
in particular, linkage of cellular membranes with the underlying cytoskeleton via cytoskeletal linker proteins helps maintain the specialized cellular arrangements necessary to glial and neuronal function.
the potential of whrn to link plasma membrane proteins with multiple cytoskeletal protein partners has been well established
<cit> , yet a precise role for whrn in the central or peripheral nervous system or even more specifically in myelinated neurons has not been examined.
in myelinated axons, axonal membrane proteins like caspr and caspr <dig> help stabilize domain organization through linkage of  <dig> b to the underlying cytoskeleton.
this loss of organization at both the light and electron microscopy level is readily apparent in mutant mice lacking caspr
<cit> , caspr2
<cit> , and  <dig> b
<cit> .
here we report that whrn knockout animals reveal defective cytoskeletal organization and accumulation of organelles in the myelinated fibers.
our phenotypic analyses of whrn knockout mice demonstrate that loss of whrn disrupts proper paranodal compaction and long-term stability of the myelinated axons throughout development.
whrn alternative splicing and phenotypes
to understand the function of whrn in the nervous system, one must be considerate of whrn mrna splice variants and rule out confounding mouse genetic strain differences.
currently, two mutant mouse strains exist for whirlin: the whirler  mouse has a spontaneous genomic deletion of exons 6– <dig> while the whrn  knockout mouse
<cit>  has a targeted exon  <dig> deletion.
we examined the localization of several myelinated axon markers  in whrn wild-type mice from each background strain and observed no disruptions in the localization of these proteins or the morphology of the paranodes .
our initial studies comparing whrn−/− to whrnwi/wi mouse strains suggested myelinated domain organization, early paranodal disorganization, and nerve conduction  was indistinguishable between each mutant mouse line.
given the novelty of the phenotype, we additionally substantiated no mouse strain effects after back-crossing to isogenic c57bl <dig> mice for two generations.
given the consistency of the mutant phenotype regardless of background mouse genetics, we were assured the phenotype was attributed to loss of whrn function and not alternative whrn splice variants or mouse genetic background variation.
scaffolding by whrn and previously established protein networks may underlie paranodal compaction and stabilization
whrn has several established roles derived from its complex and numerous interactions with other protein partners within the ear and eye.
this complexity comes from identifying and comparing the human and mouse whrn mutants and splice variants.
the significant bulk of whrn research has been performed in the ear and eye since human whrn mutations contribute to a subset of usher syndromes.
in the eye, full-length whrn colocalizes with the transmembrane proteins usherin and vlgr <dig> at the periciliary membrane complex in photoreceptors
<cit> .
studies demonstrate the two n-terminal pdz domains of the full-length whrn isoform are responsible for this interaction.
in the ear, the shorter whrn isoform has a more significant role since mutation and/or loss of whrn’s c-terminus correlates to poorer hearing deficits compared to whrn’s n-terminus.
the whrn short isoform interacts through its proline-rich domain and last pdz domain with myosin xva and mpp1/p <dig>  additional reports demonstrate whrn protein expression in the mouse cerebrum, cerebellum, and brainstem
<cit> , rat cerebellum
<cit> , and in the drosophila central nervous system
<cit> .
taken together, whrn has a well-documented history of shared protein-protein interaction across multiple model systems and cells.
we propose that whrn as a cytoskeletal scaffold crosslinks a subcellular axonal meshwork to stabilize the paranode, juxtaparanode, or both, and that in the absence of whrn, subcellular compaction of the paranode and organization of underlying microtubules, neurofilaments, cause cytoskeletal disorganization leading to an accumulation of mitochondria, and lipid vesicles along myelinated axons.
here we propose the potential sites of interaction for whrn within these regions given its established protein-protein interactions in vivo.
within the paranode, the intracellular c-terminus of caspr contains a sh <dig> domain, a potential site of interaction with whrn’s proline-rich domain.
the paranode and juxtaparanode are also enriched for  <dig> b, a known protein partner in the ear stereocilia for mpp1/p <dig> which interacts with whrn
<cit> .
caspr <dig> also contains a pdz-binding motif which could potentially interact with one of whrn’s pdz domains
<cit> .
finally, the c-terminus of whrn has the potential for self-oligomerization
<cit> , allowing for even more complex networks of protein-protein interaction within the myelinated axon.
domain organization in myelinated axons is a complicated developmental process, culminating from intrinsic and extrinsic cellular factors.
here we demonstrate that whrn expression is important for proper axonal domain organization and expand the role of whrn outside the ear and eye.
the phenotypes observed in the myelinated axons highlight that the paranodal-juxtaparanodal interface represents a substantial region for insight into paranodal stabilization and potential interaction with the axonal cytoskeleton.
in light of recent evidence of  <dig> g’s role at the internode and mesaxon, this paranodal-juxtaparanodal interface may represent an important subdomain in the study of myelinated fibers.
whrn’s protein domains have the potential to stabilize the paranodal myelin loops and associated cytoskeleton through direct or indirect interactions with caspr,  <dig> b, or other unidentified cytoskeletal proteins.
these observations are correlated using several techniques including biochemistry, light and electron microscopy.
our observed paranodal phenotypes are consistent throughout development  and similar between central and peripheral nervous systems.
one final important consideration about cytoskeletal linker proteins, both whrn and  <dig> b null mice have no statistical difference in conduction velocities in sciatic nerves compared to wild-type mice, despite having clear paranodal instability in caspr-stained myelinated fibers.
such data suggest cytoskeletal linker proteins may be functionally redundant with respect to myelinated domain organization and may require secondary or tertiary genetic ablations to achieve any measurable electrophysiological effects.
to this point, we observed the increase in purkinje axonal swellings in the double whrn;  <dig> b null mouse cerebellum compared to the single whrn knockout or wild-type mouse cerebellum.
in conclusion, our observations indicate whrn acts as a cytoskeletal scaffolding protein that is essential for proper paranodal compaction and stabilization of the axonal cytoskeleton for long-term health of myelinated axons.

 nervous system function depends on proper molecular organization between neurons and glial cells. in myelinated neurons, the segregation and enrichment of proteins in the defined domains, the node of ranvier, paranode, and juxtaparanode, is critical for saltatory action potential propagation. without proper organization, electrical nerve conduction is delayed and can result in significant motor and sensory deficits. the paranodal domain is a region of direct interaction between the glial myelin membrane loops and neuronal plasma membrane. axo-glial septate junctions link these glial membrane loops to the axonal membrane and establish the paranodal ionic barrier separating sodium channel-enriched node from the potassium channel-enriched juxtaparanode. agsj’s are composed of three transmembrane proteins: contactin, contactin-associated protein, and neurofascin. genetic ablation of any of the three molecules disrupts the paranodal barrier function and results in degraded action potential propagation. while much research has focused on these three proteins, other molecules contribute to domain formation and stabilization. for example, caspr and tag- are required for the organization of the juxtaparanodal domain and localization of potassium channels. for long-term stability, caspr and caspr are thought to rely on cytoskeletal scaffold proteins to link their associated complexes with the axonal cytoskeleton. loss of caspr results in significant cytoskeletal disorder, and since its extracellular partner cont lacks an intracellular c-terminus, the c-terminus of caspr is likely a critical region of interaction between agsj’s and the axonal cytoskeleton. previous studies using b mutant mice revealed that the b cytoskeletal protein contributes to paranodal and juxtaparanodal stability. a recent report also highlights g as an organizer of internodes in the peripheral nervous system. the neuron is abundant with cytoskeletal scaffolds however, and such findings do not exclude the possibility that other cytoskeletal elements may contribute to paranodal maintenance and long-term stability. whrn is a cytoskeletal scaffold protein that plays an important role in vision and hearing. in humans, whirlin mutations have been linked to usher syndrome type ii, an autosomal recessive vision-hearing impairment disorder. in mice, the whrn coding sequence consists of exons with two dominant splice variants, a full length and short isoform. both variants contain a proline-rich domain and pdz-domain which have been shown to link submembranous cytoskeletal elements to transmembrane complexes, as well as to self-oligomerize. in the eye, whrn interacts with the transmembrane proteins usherin and very large g protein-coupled receptor- within the periciliary membrane complex of photoreceptors. likewise in the inner ear, whrn interacts with usherin and vlgr1/gpr which form the stereociliary ankle-links. additionally, whrn interacts indirectly with b and r through mpp1/p55, and directly with myosin xva. finally, whrn is implicated with esp in stereocilia-length regulation. while the function of whrn in the ear and the eye has received significant attention, very little is known about its function in the central and/or peripheral nervous system. there are reports of whrn protein expression in the cerebrum, cerebellum, and brainstem in wild-type mice and the protein is absent in whrn knockout and whirler mutant mice. in drosophila, the closest homolog to whrn is dyschronic. in dysc mutants there is arrhythmic locomotor behavior but the eclosion circadian rhythms and clock protein cycling is unaffected. here we report that whrn is involved in proper compaction of the paranodal region in myelinated axons and for proper stabilization of the axonal cytoskeleton.  whrn is expressed in central and peripheral nervous system tissues throughout development whrn is a cytoskeletal scaffolding protein which functions to link membrane protein complexes to the cytoskeleton within hair cell stereocilia of the ear and photoreceptors in the eye. to begin assessing its function in myelinated neurons, we obtained whrn exon knockout mice. as reported previously, the murine whrn locus consists of exons with two dominant splice variants, a full length isoform and a short isoform. both variants contain pdz-domains and a proline-rich domain. after initial back crossing to c57bl mouse strain, we identified and confirmed the whrn genotype using pcr methods. to begin characterizing mrna expression of whrn in the central and peripheral nervous systems, we examined the relative expression of whrn by reverse transcriptase polymerase chain reaction in dorsal root ganglia, sciatic nerves, and spinal cord tissues in postnatal 21 day-old mice. with this subset of tissues we could delineate the origin of whrn expression as sc tissue is a combination of glial and neuronal nuclei, drg is predominantly neuronal, and sn is principally glial cytoplasm and nuclei. since whrn has two major isoforms, we designed specific primers to distinguish the full length isoform alone and those common to the full length and short isoforms. no expression of whrn isoforms was observed in whrn−/− mice. robust expression of whrn mrna was observed in drg and sc tissue while weak expression was observed in sn tissue. interestingly, weak expression of whrn mrna was observed in drg and sc tissue but no significant expression was observed in sn tissue. actin mrna expression was used as a control for total rna present. relative expression was quantified as a ratio of whrn to actin mrna between three total rt-pcr analyses. after finding whrn mrna expression in the wild-type cns and pns neuronal tissues, we next sought to determine its protein expression in p whrn+/+ versus whrn−/− mice by immunoblot analysis. in order to pursue these experiments, we generated several antibodies and affinity-purified one to the non-domain encoding, c-terminal region of whrn. after affinity-purification, wild-type and whrn knockout lysates derived from drg, sn, sc, and whole eye were immunoblotted. whole eye lysates were used as a positive control for protein expression based on previous reports. a 110 kda whrn band representing the full length protein was present in drg, sc, and whole eye lysates. no 110 kda whrn band was observed in whrn−/− lysates or in wild-type sn lysate. each tissue type showed similar total protein levels between genotypes based on total tubulin on immunoblots. having confirmed whrn−/− tissues were deficient in whrn mrna and protein, we next sought to determine if loss of whrn resulted in altered protein expression of known nodal, paranodal, and juxtaparanodal proteins. we prepared spinal cord lysates from and 16 week-old wild-type and whrn knockout mice and immunoblotted for whrn. whrn protein expression levels were similar from to 16 weeks in wild-type mice while no expression was observed in the whrn−/− at any time point. next, we immunoblotted for the paranodal protein caspr and found similar expression levels between 4–16 week-old in both whrn+/+ and whrn−/− mice. using a pan-neurofascin-c-terminal antibody, we observed similar levels of nodal neurofascin and paranodal neurofascin protein expression in wild-type and whrn knockout mice across 4–16 weeks of age. examination of neuronal cytoskeletal protein b and juxtaparanodal protein caspr shows steady protein expression levels in wild-type and whrn knockout mice from 4–16 weeks of age. finally, previous reports showed in vivo interaction between rat whrn/cip and calmodulin-dependent serine kinase, a synaptic organization protein. after immunoblotting for cask, no difference in its protein level was observed across whrn genotype or ages from 4–16 weeks. total protein levels in spinal cord lysates were consistent across the ages and genotypes based on tubulin levels. in summary, these developmental expression profiles suggest that loss of whrn expression does not affect the overall expression or stability of other axonal domain markers. 1b expressed as a ratio of whrn to actin band intensity. d. immunoblots of 110 kda whrn protein band in wild-type and whrn mutant drg, sn, sc, and whole eye using affinity-purified rbwhrn antibody. αtubulin served as a loading control. e. immunoblots of wild-type and whrn knockout mutant and 16-week-old spinal cord lysates. the expression profile using various myelinated axonal domain markers includes caspr, neurofascin, b, caspr as well as cask. αtubulin served as a loading control. f. immunofluorescence of teased sciatic nerve fibers from wild-type and whrn knockout mice mice. neurofascin and paranodal caspr reveal paranodal compaction is disrupted in whrn knockout fibers. note nfct detects both paranodal nf and nodal nf isoforms. whrn knockout mice reveal a quantifiable paranodal compaction phenotype in peripheral myelinated axons to determine the effects of whrn loss on axonal domain organization, we examined well-characterized nodal, paranodal, and juxtaparanodal markers by immunofluorescence in wild-type and whrn−/− mice. extensive and repeated immunostaining with our whrn antibody revealed no consistent localization or differences in whrn localization between wild-type to whrn−/− fibers. compared to wild-type, the most striking observation in teased whrn−/− sciatic nerve fibers was the spring-like separation of the paranodal axo-glial septate junction loops beginning along the paranodal-juxtaparanodal border as observed by caspr and nf immunostaining. irregular paranodal compaction is rarely observed in wild-type fibers, so we sought to quantify the overall observation of this phenotype in whrn+/+ and whrn−/− fibers. we utilized a blinded counting strategy to count spring-like phenotypes from wild-type or whrn−/− caspr-stained paranodes. a statistically significant difference was observed in the percentage of irregular paranodal compaction at % in wild-type fibers compared to % in whrn knockout mice. having confirmed the significance of this spring-like paranodal phenotype in 7 week-old whrn knockout fibers, we expanded our analysis to 4–8 week-old time points. sciatic nerves from whrn+/+ or whrn−/− littermate mice at and 8 weeks, were immunostained with antibodies against nodal, paranodal, and juxtaparanodal markers. in the 4 week-old wild-type sciatic nerve, juxtaparanodal kv was separated from nodal nf by paranodal caspr. this demarcation of domains in myelinated fibers is also observed in 4 week-old whrn−/− fibers. myelin loops of agsjs in 4 week-old whrn−/− sciatic nerve fibers were already separating away from the paranode compared to 4 week-old wild-type sciatic nerve fibers. breaks in kv localization were observed even where caspr staining was limited to a single myelin loop, suggesting agsj’s barrier function was conserved. juxtaparanodal localization was diffuse and asymmetric in whrn−/− fibers compared to wild-type. when comparing wild-type to whrn knockout mice, we observed no obvious differences in intensity levels of juxtaparanodal or nodal markers. at 6 weeks, teased wild-type sciatic nerves showed typical domain organization. 6 week-old whrn null sciatic nerves have typical nf nodal domain organization while caspr paranodal compaction and kv juxtaparanodal localization were disrupted. additional immunostaining in 6 week-old whrn null sciatic nerves with a pan-neurofascin-c-terminal antibody detected both nodal nf and paranodal nf paranodal nf and caspr immunostaining mostly colocalize in whrn−/− sciatic nerve fibers and revealed similar problems with paranodal compaction. ankg nodal domain organization in 6 week-old whrn−/− sciatic nerve fibers appeared similar to wild-type. finally, immunostained 8 week-old sciatic nerves revealed similar nf nodal and ankg nodal organization. like and 6 week-old sciatic nerves, caspr and nfct immunostaining consistently revealed paranodal compaction defects as well as kv juxtaparanodal diffusion in whrn knockout mice when compared with wild-type. to determine if such peripheral nerve phenotypes could be the result of differences in inner mesaxons, we immunostaining 7 week-old wild-type and whrn knockout fibers with mag but observed no striking phenotypic differences in localization. in summary, the paranodal and juxtaparanodal regions displayed phenotypes that suggest that normal compaction of the paranodal loops fails to occur in whrn knockout mice at and 8 weeks of age. during initial quantification we observed that larger diameter myelinated fibers had proportionally more paranode compaction defects compared to thinner caliber myelinated fibers, so we imaged and assembled twenty caspr-stained confocal images for each genotype and time point to assess any subtle, sub-micron paranodal changes by light microscopy due to whrn loss. next, we measured various dimensions of the paranode. note images collected were from ~ micron caliber myelinated neurons. using zeiss software, micron measurements were recorded, tabulated, and reported as averages with standard deviations for the nodal gap, the paranodal diameter, paranodal width, as well as the percentage of paranodes which display paranodal compaction abnormalities. the nodal gap for wild-type and whrn−/− sciatic nerves was ±  μm and ±  μm at 4 weeks, ±  μm and ±  μm at 6 weeks, and ±  μm and ± at 8 weeks respectively. no statistically significant difference was noted between ages or genotypes with respect to nodal gap. the paranodal diameter for wild-type and whrn−/− sciatic nerves was ±  μm and ±  μm at 4 weeks, ±  μm and ±  μm at 6 weeks, and ±  μm and ± at 8 weeks respectively. with respect to paranodal diameter, measurements were similar between all groups. the paranodal width for wild-type and whrn−/− sciatic nerves was ±  μm and ±  μm at 4 weeks, ±  μm and ±  μm at 6 weeks, and ±  μm and ± at 8 weeks respectively. no statistically significant difference was found in paranodal width. the paranodal compaction phenotype percentage for wild-type and whrn−/− sciatic nerves was 15% and 55% at 4 weeks, 15% and 50% at 6 weeks, and 15% and 55% respectively. as a reminder, the increase in the whrn knockout phenotype percentage represents the shift in selection from all fibers to larger-caliber myelinated fibers. note the greater percentage of paranodes with compaction issues in whrn−/− sciatic nerves likely contributes to the increased deviation in paranodal widths. additionally, comparisons between 8 week-old wild-type and whrn−/− sciatic nerve fibers demonstrated no significant difference in the conduction velocity or waveforms of measured compound action potentials in two separate measurement trials. whrn knockout mouse sciatic nerve and spinal cord myelinated fibers display paranodal compaction abnormalities throughout development to expand on the characterization of whrn loss with respect to myelinated domain organization, we examined a larger developmental window from postnatal ages 2 weeks to 1 year. wild-type and whrn knockout sciatic nerves revealed the following percentages of caspr-stained phenotype-positive paranodes: % and % at 10 weeks, % and % at 20 weeks, % and % at 30 weeks, % and % at 40 weeks, and % and % at 1 year, respectively. similar to the 4–8 week studies, we immunostained fibers with nodal, paranodal, and juxtaparanodal markers, as well as the axonal cytoskeletal protein markers b and heavy chain neurofilament given whrn’s known cytoskeletal scaffolding role. we observed no differences in nodal formation using nf in 2 week-old fibers. abnormalities in paranodal formation and com-paction were observed in caspr-stained whrn−/− fibers compared to wild-type. overall kv juxtaparanodal signal appeared similar between 2 week-old wild-type and whrn−/− fibers with accumulation and enrichment of kv channels neighboring caspr within the internodal region. no obvious difference in intensity or localization of nfl-h or b was found in 2 week-old sciatic nerve fibers between whrn genotypes. with 10 week-old sciatic nerve fibers, we observed no differences in nodal domains using nf. like 8 week-old fibers, we observed 10 week-old whrn−/− fibers with abnormal paranodal compaction, when compared to wild-type fibers. juxtaparanodal domains stained with kv appear more diffuse but similar in overall intensity in 10 week-old whrn−/− fibers when compared to wild-type. we observed slight enrichment of nfl-h and b within the paranodal region of 10 week-old wild-type sciatic nerves compared to whrn knockout mice, but the overall intensity of nfl-h and b along the remaining axon appeared similar. immunostaining of and 40 week-old wild-type sciatic nerve fibers were performed and domain organization was identical to 10 week-old wild-type fibers. looking at and 40 week-old whrn−/− peripheral nerve fibers, we observed no differences in nodal domains between whrn wild-types or knockout mice using nf. similar to 10 week-old fibers, we observed numerous and 40 week-old whrn null fibers stained with caspr with abnormal paranodal compaction when compared to wild-type fibers. in and 40-week old whrn−/− fibers, juxtaparanodal domains stained with kv showed numerous breaks at sites of caspr signal, appeared less symmetrical, and displayed similar overall signal when compared with wild-type. cytoskeletal marker nfl-h staining was generally uniform within the axon, and b was present in all axonal domains except the node in and 40 week-old whrn−/− sciatic nerves. one-year-old sciatic nerves showed no difference in nodal organization. one year-old whrn−/− sciatic nerves revealed abnormal paranodal compaction compared to wild-type fibers. notably, we observed paranodal compaction defects in older whrn−/− sciatic nerve fibers like blocks and bulges in caspr signal as well as the spring-like phenotype observed in younger fibers. as seen previously, the juxtaparanodal domains of whrn−/− peripheral nerves stained with kv showed significant asymmetric enrichment and periodic breaks as well as overlap with caspr signal when compared with one year-old wild-type fibers. also, patchy cytoskeletal-deficient staining was observed in older whrn−/− fibers near the paranode. cytoskeletal marker nfl-h staining was weakly enriched within the paranode of the wild-type axon and less uniform within whrn−/− fibers with paranodal compaction problems. likewise, b showed disrupted juxtaparanodal cytoskeletal staining in one year-old whrn−/− fibers which was not observed in the wild-type fibers. we also sought to examine the effects of whrn loss on axonal domain organization in the central nervous system. utilizing white matter tracts in the spinal cord, we were able to identify by immunostaining subtle but consistent differences in paranodal compaction. as in the pns, we stained longitudinal spinal cord sections with nodal, paranodal, and juxtaparanodal markers. wild-type and whrn knockout spinal cord fibers revealed the following percentages of caspr-stained phenotype-positive paranodes: % and % at 10 weeks, % and % at 20 weeks, % and % at 30 weeks, and % and % at 40 weeks. no differences in nodal organization were observed in 2 week-old spinal cord sections. at 40 week-old, nodal organization appeared similar between wild-type and whrn−/− spinal cord fibers using nf. immunostaining with paranodal caspr revealed subtle and infrequent paranodal abnormalities in 2 week-old whrn null spinal cord fibers compared to wild-type. in contrast, obvious and regular paranodal compaction defects were observed in,,, and 40 week-old whrn−/− spinal cords compared to their wild-type controls. finally, juxtaparanodal kv immunostaining of,,, and 40 week-old wild-type spinal cord myelinated axons showed similar overall expression as whrn knockout axons, but consistent juxtaparanodal disorganization and colocalization with loosened caspr-stained myelin loops was observed most often in whrn−/− fibers. in summary, there is significant evidence that loss of whrn disrupts normal paranodal compaction in 10–40 week-old myelinated spinal cord axons with subsequent effects on juxtaparanodal organization. taken together, both the peripheral and central nervous systems likely utilize the cytoskeletal properties of whrn to help stabilize the cellular organization between myelinating glial cells and neurons around the paranodal region throughout development. whirlin knockout mice have cerebellar purkinje cells with bead-like, axonal swellings to determine the effects of whrn loss on cerebellar purkinje cell morphology, we immunostained cerebellar slices from 6 week-old wild-type, whrn knockout, and double whrn and b null animals. given that caspr and cgt, two genes critical for formation of a proper paranode, display purkinje axonal swellings and cytoskeletal disorganization, we were curious as to the effects of the combined loss of whrn and b. while no obvious differences in localization was observed using our whrn antibody in any of these genotypes, we immunostained against b, purkinje-specific calbindin, and glial-specific myelin basic protein/mbp. there was no difference in b staining intensity between wild-type and whrn knockout slices. upon examining whrn−/− cerebellum sections, purkinje axons appeared to contain bead-like, swellings along their extensions through the granular layer using both calb and mbp in comparison to a uniform caliber axon in wild-type fibers. the secondary loss of b protein resulted in more swellings observed with calb and mbp when compared with whrn null animals alone. in summary, the cytoskeletal elements whrn and b likely have an assistive role in preventing cytoskeletal accumulation and disorganization within cerebellar purkinje cell axons in a similar phenotypic manner to caspr null mice. ultrastructural abnormalities in whrn knockout sciatic nerve, spinal cord fibers, and cerebellar purkinje axons to further understand whrn’s role in myelinated axons, transmission electron microscopy was performed in order to examine the ultrastructural architecture in myelinated axons in the pns and cns of 7 week-old and 3 month-old wild-type and whrn−/− mice. low-magnification, cross-section electron micrographs of wild-type and whrn knockout myelinated sciatic nerve fibers showed the typical organization of tightly bound, electron-dense myelin wraps around the internodal region of the axonal membrane. accumulation of mitochondria and lipid vesicles in the internodal regions was clearly observed in whrn knockout animals compared to wild-type sciatic nerve fibers. given the potential role of the mesaxon in the observed light microscope phenotype, we found no striking differences in the ultrastructural organization or arrangements of the inner mesaxon along the internodal region at 7 weeks or 3 months of age. consistent with our immunostaining data, no obvious differences in nodal organization were observed in either genotype. higher magnification along the wild-type paranodal region revealed the hallmark electron-dense agsjs formed between the glial paranodal loops and axolemma and accompanying parallel arrays of cytoskeletal elements in the axon. in contrast, the whrn knockout paranodal region of 7 week-old and 3 month-old displayed poorly defined but present agsjs, less organized neurofilaments and microtubules, and consistent accumulation of mitochondria and lipid vesicles in the paranodal region. the ultrastructural phenotypes displayed at both 7 weeks and 3 months of age suggest that whrn is important for the long-term maintenance and the overall structure of the myelinated axons. in the central nervous system, low-magnification, electron micrographs of wild-type and whrn knockout myelinated fibers showed slight differences in spinal cord cross-sections. like the peripheral nerves, the overall organization between glial cell and neuron remained similar between wild-type and whrn−/− fibers. however, mitochondria were slightly more abundant in whrn knockout fibers. no obvious ultrastructural differences were observed in the node of either genotype, but greater accumulation of mitochondria and lipid vesicles was observed in the paranodal and juxtaparanodal regions. higher magnification along the paranodal region revealed the expected electron-dense agsjs formed between myelin loops and the axolemma. like in the pns, the whrn knockout paranodal region of 7 week-old and 3 month-old displayed poorly defined but present agsjs and accumulation of mitochondria and lipid vesicles in the paranodal region. examining the cerebellum, we observed purkinje axon swellings in whrn knockout fibers. low-magnification electron micrographs of 7 week-old wild-type and whrn knockout at 7 weeks and 3 months of age showed striking differences in purkinje axon myelinated fibers in the cerebellum granular layer. this region shows axonal swellings filled with densely-packed organelles, particularly mitochondria and vesicles. taken together, the ultrastructural analyses of whrn knockout mice demonstrate that whrn is critical for the stability of paranodal organization, proper axonal cytoskeletal arrangements, and prevention of sub-cellular organelle accumulation in myelinated axons.  cellular and molecular interactions between neurons and glia establish and stratify the numerous tasks of the nervous system. in particular, linkage of cellular membranes with the underlying cytoskeleton via cytoskeletal linker proteins helps maintain the specialized cellular arrangements necessary to glial and neuronal function. the potential of whrn to link plasma membrane proteins with multiple cytoskeletal protein partners has been well established, yet a precise role for whrn in the central or peripheral nervous system or even more specifically in myelinated neurons has not been examined. in myelinated axons, axonal membrane proteins like caspr and caspr help stabilize domain organization through linkage of b to the underlying cytoskeleton. this loss of organization at both the light and electron microscopy level is readily apparent in mutant mice lacking caspr, caspr2, and b. here we report that whrn knockout animals reveal defective cytoskeletal organization and accumulation of organelles in the myelinated fibers. our phenotypic analyses of whrn knockout mice demonstrate that loss of whrn disrupts proper paranodal compaction and long-term stability of the myelinated axons throughout development. whrn alternative splicing and phenotypes to understand the function of whrn in the nervous system, one must be considerate of whrn mrna splice variants and rule out confounding mouse genetic strain differences. currently, two mutant mouse strains exist for whirlin: the whirler mouse has a spontaneous genomic deletion of exons 6– while the whrn knockout mouse has a targeted exon deletion. we examined the localization of several myelinated axon markers in whrn wild-type mice from each background strain and observed no disruptions in the localization of these proteins or the morphology of the paranodes. our initial studies comparing whrn−/− to whrnwi/wi mouse strains suggested myelinated domain organization, early paranodal disorganization, and nerve conduction was indistinguishable between each mutant mouse line. given the novelty of the phenotype, we additionally substantiated no mouse strain effects after back-crossing to isogenic c57bl mice for two generations. given the consistency of the mutant phenotype regardless of background mouse genetics, we were assured the phenotype was attributed to loss of whrn function and not alternative whrn splice variants or mouse genetic background variation. scaffolding by whrn and previously established protein networks may underlie paranodal compaction and stabilization whrn has several established roles derived from its complex and numerous interactions with other protein partners within the ear and eye. this complexity comes from identifying and comparing the human and mouse whrn mutants and splice variants. the significant bulk of whrn research has been performed in the ear and eye since human whrn mutations contribute to a subset of usher syndromes. in the eye, full-length whrn colocalizes with the transmembrane proteins usherin and vlgr at the periciliary membrane complex in photoreceptors. studies demonstrate the two n-terminal pdz domains of the full-length whrn isoform are responsible for this interaction. in the ear, the shorter whrn isoform has a more significant role since mutation and/or loss of whrn’s c-terminus correlates to poorer hearing deficits compared to whrn’s n-terminus. the whrn short isoform interacts through its proline-rich domain and last pdz domain with myosin xva and mpp1/p additional reports demonstrate whrn protein expression in the mouse cerebrum, cerebellum, and brainstem, rat cerebellum, and in the drosophila central nervous system. taken together, whrn has a well-documented history of shared protein-protein interaction across multiple model systems and cells. we propose that whrn as a cytoskeletal scaffold crosslinks a subcellular axonal meshwork to stabilize the paranode, juxtaparanode, or both, and that in the absence of whrn, subcellular compaction of the paranode and organization of underlying microtubules, neurofilaments, cause cytoskeletal disorganization leading to an accumulation of mitochondria, and lipid vesicles along myelinated axons. here we propose the potential sites of interaction for whrn within these regions given its established protein-protein interactions in vivo. within the paranode, the intracellular c-terminus of caspr contains a sh domain, a potential site of interaction with whrn’s proline-rich domain. the paranode and juxtaparanode are also enriched for b, a known protein partner in the ear stereocilia for mpp1/p which interacts with whrn. caspr also contains a pdz-binding motif which could potentially interact with one of whrn’s pdz domains. finally, the c-terminus of whrn has the potential for self-oligomerization, allowing for even more complex networks of protein-protein interaction within the myelinated axon. domain organization in myelinated axons is a complicated developmental process, culminating from intrinsic and extrinsic cellular factors. here we demonstrate that whrn expression is important for proper axonal domain organization and expand the role of whrn outside the ear and eye. the phenotypes observed in the myelinated axons highlight that the paranodal-juxtaparanodal interface represents a substantial region for insight into paranodal stabilization and potential interaction with the axonal cytoskeleton. in light of recent evidence of g’s role at the internode and mesaxon, this paranodal-juxtaparanodal interface may represent an important subdomain in the study of myelinated fibers. whrn’s protein domains have the potential to stabilize the paranodal myelin loops and associated cytoskeleton through direct or indirect interactions with caspr, b, or other unidentified cytoskeletal proteins. these observations are correlated using several techniques including biochemistry, light and electron microscopy. our observed paranodal phenotypes are consistent throughout development and similar between central and peripheral nervous systems. one final important consideration about cytoskeletal linker proteins, both whrn and b null mice have no statistical difference in conduction velocities in sciatic nerves compared to wild-type mice, despite having clear paranodal instability in caspr-stained myelinated fibers. such data suggest cytoskeletal linker proteins may be functionally redundant with respect to myelinated domain organization and may require secondary or tertiary genetic ablations to achieve any measurable electrophysiological effects. to this point, we observed the increase in purkinje axonal swellings in the double whrn; b null mouse cerebellum compared to the single whrn knockout or wild-type mouse cerebellum. in conclusion, our observations indicate whrn acts as a cytoskeletal scaffolding protein that is essential for proper paranodal compaction and stabilization of the axonal cytoskeleton for long-term health of myelinated axons. 
light and electron microscopic analyses of whrn knockout mice reveal bead-like swellings in cerebellar purkinje axons containing mitochondria and vesicles by both. whrn likely contributes to the stabilization of paranodal myelin loops and axonal cytoskeleton through yet unconfirmed cytoskeletal proteins. a significant lack of knowledge remains as to cytoskeletal proteins which stabilize paranodal domains and underlying cytoskeleton. here we demonstrate that loss of whrn disrupts proper axonal domain organization. in conclusion, our observations suggest that whrn is not required for the organization of axonal domains, but once organized, whrn acts as a cytoskeletal linker to ensure proper paranodal compaction and stabilization of the axonal cytoskeleton in myelinated axons. without proper organization, electrical nerve conduction is delayed, resulting in detrimental physiological outcomes. comparing wild-type mice to whrn knockout mice, we observed no significant differences in the expression of standard axonal domain markers by immunoblot analysis but observed and quantified a novel paranodal compaction phenotype in to 8 week-old whrn−/− nerves. whirlin is a pdz domain-containing cytoskeletal scaffold whose absence in humans results in usher syndromes or variable deafness-blindness syndromes. domain organization in myelinated axons remains a complex developmental process. rt-pcr and immunoblot analyses revealed expression of whrn mrna and whrn full-length protein, respectively, in several stages of central and peripheral nervous system development. the paranodal compaction phenotype and associated cytoskeletal disruption was observed in whrn−/− mutant sciatic nerves and spinal cord fibers from early to late stages of development. paranodal abnormalities are consistently observed throughout development and similar between central and peripheral nervous systems.
 myelinated axons are organized into distinct subcellular and molecular regions. without proper organization, electrical nerve conduction is delayed, resulting in detrimental physiological outcomes. one such region is the paranode where axo-glial septate junctions act as a molecular fence to separate the sodium channel-enriched node from the potassium channel-enriched juxtaparanode. a significant lack of knowledge remains as to cytoskeletal proteins which stabilize paranodal domains and underlying cytoskeleton. whirlin is a pdz domain-containing cytoskeletal scaffold whose absence in humans results in usher syndromes or variable deafness-blindness syndromes. mutant whirlin mouse model studies have linked such behavioral deficits to improper localization of critical transmembrane protein complexes in the ear and eye. until now, no reports exist about the function of whrn in myelinated axons.  rt-pcr and immunoblot analyses revealed expression of whrn mrna and whrn full-length protein, respectively, in several stages of central and peripheral nervous system development. comparing wild-type mice to whrn knockout mice, we observed no significant differences in the expression of standard axonal domain markers by immunoblot analysis but observed and quantified a novel paranodal compaction phenotype in to 8 week-old whrn−/− nerves. the paranodal compaction phenotype and associated cytoskeletal disruption was observed in whrn−/− mutant sciatic nerves and spinal cord fibers from early to late stages of development. light and electron microscopic analyses of whrn knockout mice reveal bead-like swellings in cerebellar purkinje axons containing mitochondria and vesicles by both. these data suggest that whrn plays a role in proper cytoskeletal organization in myelinated axons.  domain organization in myelinated axons remains a complex developmental process. here we demonstrate that loss of whrn disrupts proper axonal domain organization. whrn likely contributes to the stabilization of paranodal myelin loops and axonal cytoskeleton through yet unconfirmed cytoskeletal proteins. paranodal abnormalities are consistently observed throughout development and similar between central and peripheral nervous systems. in conclusion, our observations suggest that whrn is not required for the organization of axonal domains, but once organized, whrn acts as a cytoskeletal linker to ensure proper paranodal compaction and stabilization of the axonal cytoskeleton in myelinated axons. myelinated axonsaxonal domainsparanodal domainaxonal cytoskeletonwhirlin 