Ultrastructural Abnormalities in Whrn Knockout

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 (Fig. 3.6) and CNS (Fig. 3.7) of three month-old wild-type and Whrn-/- mice. Low- magnification, cross-section electron micrographs of wild-type (Fig. 3.6A) and Whrn knockout (Fig. 3.6B) 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 seen more often in Whrn knockout compared to wild-type sciatic nerve fibers (Fig. 3.6B,C vs. 3.6A,D, black arrowheads). Consistent with our immunostaining data, no obvious differences in nodal organization was observed in either genotype (data not shown). Higher magnification along the wild-type paranodal region (Fig. 3.6E, small black arrowheads) 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 (Fig. 3.6F, small black arrowheads) displayed poorly defined but present AGSJs, less organized neurofilaments and microtubules, and consistent accumulation of mitochondria and lipid vesicles (Fig. 3.6F, large black arrowheads) in the paranodal region. Similarly, the paranodal-juxtaparanodal transition region of the myelinated axons showed further accumulation of mitochondria and lipid vesicles and cytoskeletal disorganization in Whrn-/- tissue (Fig. 3.6H, black arrowheads) compared to

103

Figure 3.6. Ultrastructural examination of three month-old Whirlin knockout sciatic nerves reveals organelle accumulation and cytoskeletal disruption.

A–B, Low magnification electron micrographs through the internodal regions of sciatic nerves in three month-old wild-type and Whrn knockout mice in cross section (A vs. B) and longitudinal orientations (C vs. D). Overall cellular organization between Whrn knockout and wild-type sciatic nerve fibers is conserved with tightly compacted myelin around each axon. At a higher magnification, the wild-type paranodal loops (E, small arrowheads) have clearly defined characteristic transverse, electron-dense septa and parallel arrays of

cytoskeletal elements. In contrast, Whrn mutant paranodal septa (F, small arrowheads) are less definitive and fuzzy with associated accumulation of organelles. Similarly, the

paranodal-juxtaparanodal axonal region shows similar accumulation of organelles, particularly mitochondria and transport vesicles, in Whrn knockout animals (H, large arrowheads) compared to wild-type (G). Scale bars: A-B: 2μm, C-D: 1μm; E-H, 200nm.

104

wild-type (Fig. 3.6G). These results suggest that Whrn is important for proper long-term maintenance of the overall structure of the myelinated axons.

Turning to the central nervous system, low-magnification, cross-section electron micrographs of three month-old wild-type (Fig. 3.7A) and Whrn knockout (Fig. 3.7B) fibers showed slight differences in spinal cord myelinated fibers. 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. This observation was more consistent looking at the nodal-paranodal longitudinal sections (Fig. 3.7C-D) of spinal cord sections. No obvious ultrastructural differences were observed in node of either genotype, but longitudinal electron micrographs of paranodal and

juxtaparanodal regions showed greater accumulation of mitochondria and lipid vesicles as well as microtubule and neurofilament disorganization in Whrn-/- spinal cords (Fig. 3.7D, black arrowheads) compared to wild-type fibers (Fig. 3.7C). Examining the cerebellum, we observed and confirmed Purkinje axon swellings in Whrn knockout fibers (Fig. 3.7F-H). Low-magnification electron micrographs of three month-old wild-type (Fig. 3.7E) and Whrn knockout (Fig. 3.7F-H) cerebellum granular layer showed striking differences in Purkinje axon myelinated fibers. This region shows axonal swellings filled with densely-packed organelles, particularly mitochondria and vesicles, in Whrn knockout animals (Fig. 3.7F-H, black arrowheads) compared to wild-type (Fig. 3.7E). 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.

106

Figure 3.7. Ultrastructural examination of Whirlin knockout central nervous system tissues reveals organelle accumulation in myelinated axons.

Electron micrographs of internodal cross sections (A-B) and nodal-paranodal longitudinal (C-D) regions in three month-old spinal cords from wild-type (A, C) and Whrn knockout mice (B, D). Overall cellular organization is conserved between wild-type and Whrn-/- and sciatic nerve fibers with tightly compacted myelin around each axon. Enrichment of

mitochondria and occasional myelin ruffling is observed in Whrn knockout mice compared to wild-type. E-H: Electron micrographs of cerebellar Purkinje myelinated axons running through the granular layer of three-month old mice. This region shows axonal swellings filled with densely-packed organelles, particularly mitochondria and vesicles, in Whrn knockout animals (F-H, black arrowheads) compared to wild-type (E, black arrowhead). Scale bars: A-F: 1μm.

107

3.4 Discussion

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 (Mburu, Mustapha et al. 2003, Belyantseva, Boger et al. 2005, Delprat, Michel et al. 2005, Mburu, Kikkawa et al. 2006, van Wijk, van der Zwaag et al. 2006, Okumura, Mochizuki et al. 2010, Yang, Liu et al. 2010, Manor, Disanza et al. 2011), 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 Caspr2 help stabilize domain organization through linkage of 4.1B to the underlying cytoskeleton. This loss of organization at both the light and electron microscopy level is readily apparent in mutant mice lacking Caspr (Bhat, Rios et al. 2001), Caspr2 (Poliak, Salomon et al. 2003), and 4.1B (Buttermore, Dupree et al. 2011). Here we report a milder but similar phenotype with loss of cytoskeletal organization and accumulation of organelles in the myelinated fibers of Whrn knockout animals. Comprehensive 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.