B CNPDeficient Mice

In the absence of PLP, the destabilization of the myelin sheath could be a primary cause of axon swellings and degeneration. It was important, therefore, to identify a protein that also contributes to axonal integrity, but was unrelated to the physical stability of myelin.

The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is a widely used marker protein of myelin-forming glial cells. In addition to the glial cell soma, CNP is associated with noncompacted regions of myelin such as the inner mesaxon, paranodal loops, and Schmidt-Lantermann

Figure I Axonal swelling and degeneration in mice lacking PLP/DM20. Membranous organelles accumulate preferentially distal to the nodal complex in optic nerve axons of PLP/DM20-deficient mice. (A) Schematic showing the axon and surrounding myelin sheath. The length of axon myelinated by a single oligodendrocyte process is termed the internode and terminates at the nodes of Ranvier (N). (B) The boxed area from A is shown in detail. The node of Ranvier is abutted by the paranode, the region at which the terminal loops of individual myelin lamellae (arrow 1) appose the axolemma in an orderly manner. The juxtaparanode (JPN) is the region between the paranode and the internode. The paranodal axoglial junction (arrow 2) is a highly specialized intercellular junction. (C) Wild-type axon showing a nodal complex. The node (N), paranode (P), and juxtaparanodal (J) regions are indicated, as is the distal (chiasmal) side. An axonal mitochondrion is evident (arrow). Bar 2 |m. (D) In this and subsequent images the axons are from PLP null mice. A small accumulation of dense bodies and mitochondria (arrow) are present at the distal juxtaparanode (J). The proximal juxtaparanode and internode contain several nonclustered mitochondria, which is slightly in excess of the maximum number observed in wild-type axons. However, there is a distinct difference between proximal and distal regions. Bar 2 mm. (E) In this example the accumulated dense bodies and mitochondria occupy the distal internode, juxtaparanode (J), and paranode (Pd), and have also extended into the nodal (N) region; the proximal paranode (Pp) remains unaffected. Bar 2 |m. (F) A proportion of optic nerve axons in PLP null mice have nonmyelinated regions interposed between myelinated internodes. In this example, the proximal axon (Ax) is unmyelinated, whereas the distal axon is myelinated. A heminode with its paranode (P) is present. A collection of organelles, predominantly dense bodies (arrow), is present distal to the paranode, whereas no accumulation is present proximally. Bar 2 mm. (G) The distal aspect of a nodal complex is shown with the paranode (P) marked. The distal axon contains a small accumulation of mitochondria. Bar, 1 mm. (H) Another axon shows accumulation of dense bodies distal to the paranode (P). Bar 1 |m. (Reproduced from Edgar et al., The Journal of Cell Biology 2004, 166:121-131 by copyright permission of The Rockefeller University Press.)

incisures (Trapp et al., 1988), but it is absent from compact myelin. CNP is maintained in mature oligodendrocytes throughout life. By immunoelectron microscopy, the CNP gene is also expressed at a low level outside the nervous system, including a subset of immune cells, photoreceptor cells, and in testis (Giulian and Moore, 1980; Sprinkle et al., 1985; Scherer et al., 1994).

CNP transcripts encode two protein isoforms (46 kDa and 48 kDa) (Scherer et al., 1994; O'Neill et al., 1997) that are isoprenylated at the carboxyl-terminus for efficient association with cellular membranes (Agrawal et al., 1990; Braun et al., 1991). The CNP biochemical substrates (2',3'-cyclic nucleotides) are not present in brain, leaving the enzyme activity puzzling, because they are known only as intermediates of RNA metabolism (Heaton and Eckstein, 1996). It has been suggested that CNP interacts with mitochondria and cytoskeletal proteins, and serves as a membrane anchor for tubulin (McFerran and Burgoyne, 1997; Laezza et al., 1997; Bifulco et al., 2002), although the normal cellular function of this protein remains unclear. Overexpression on CNP in transgenic mice causes myelin abormalities (Gravel et al., 1996; Yin et al., 1997). However, targeted disruption of CNP function by homologous recombination failed to unequivocally reveal the cellular function of CNP for myelination. Mutant animals show no major myelin abnormality during the first 4 months of age. The protein and lipid composition of purified myelin membranes reveal no difference, except for the lack of CNP. By electron microscopy and light microscopy, the amount and ultrastructure of CNS myelin are initially normal (Lappe-Siefke et al., 2003).

The lack of CNP from myelin, however, eventually manifests as a severe neurodegenerative disorder with premature death and similar features as described for PLP null mutants. Whereas 3-month-old CNP-deficient mice are indistinguishable in motor behavior from wild-type littermates, at about 4 months, many mutants develop ataxia and visible hind-limb impairments. At 6 months, many mutants are unable to grasp a horizontal bar and balance on top of it. Frequently, mice have convulsions, for example, when lifted at their tails. This late phenotype is associated with muscle weakness, weight loss, and kyphosis. Premature death occurs at 7 to 14 months (Lappe-Siefke et al., 2003).

On Nissl-stained sections, CNP-deficient mice reveal a reduction of overall brain size in combination with enlarged ventricles (hydrocephalus), indicating underlying neurode-generative changes (Fig. 2). Indeed, myelinated axons are visibly lost in the upper layers of the mutant cortex. Antibodies against the amyloid precursor protein reveal axonal swellings in the corpus callosum and in the lumbar spinal cord (Lappe-Siefke et al., 2003). At the electron microscopic level, axonal swellings are filled with micro-tubules, dense bodies, multivesicular bodies, and mitochondria. Myelin sheaths are generally preserved over small axonal swellings, but become attenuated through slippage

Figure 2 Neurodegeneration and hydrocephalus in mice lacking CNP. (A—E) Brain sections from CNP1 mutants and wild-type mice (age, 12 months), Nissl-stained (A) and Gallyas-impregnated to show myelin (B-E). The severely affected CNP1 mutant mouse brain (C, E) had enlarged ventricles and a reduced thickness in cortical gray and subcortical white matter (corpus callosum). Many myelinated fibers within the cortex seemed to have degenerated (arrowheads, E) compared with the equivalent layers of wild-type cortices (arrowheads, D). (F, G) PAS reaction on brain paraffin sections prepared from wild-type (F) and homozygous mutant (G) brains (corpus callosum; age, 14 mo). Many cells that accumulated PAS-positive material (arrowheads, G and inset enlargement) showed phagocytic activity, an indirect sign of neurodegeneration. Cell density was increased in this area, as the thickness of the corpus callosum was reduced. Bar 20 mm. (Reproduced from Lappe-Siefke et al., Nature Genetics 2003, 33: 366-374 by copyright permission of Nature Publishing Group.)

Figure 2 Neurodegeneration and hydrocephalus in mice lacking CNP. (A—E) Brain sections from CNP1 mutants and wild-type mice (age, 12 months), Nissl-stained (A) and Gallyas-impregnated to show myelin (B-E). The severely affected CNP1 mutant mouse brain (C, E) had enlarged ventricles and a reduced thickness in cortical gray and subcortical white matter (corpus callosum). Many myelinated fibers within the cortex seemed to have degenerated (arrowheads, E) compared with the equivalent layers of wild-type cortices (arrowheads, D). (F, G) PAS reaction on brain paraffin sections prepared from wild-type (F) and homozygous mutant (G) brains (corpus callosum; age, 14 mo). Many cells that accumulated PAS-positive material (arrowheads, G and inset enlargement) showed phagocytic activity, an indirect sign of neurodegeneration. Cell density was increased in this area, as the thickness of the corpus callosum was reduced. Bar 20 mm. (Reproduced from Lappe-Siefke et al., Nature Genetics 2003, 33: 366-374 by copyright permission of Nature Publishing Group.)

when swellings enlarge at the nodal region, similar to the situation in PLP-null mutant mice. The periodicity of myelin itself is always maintained, and its physical stability appears not to be compromised. The ultrastructure of paranodes, in which CNP is normally localized, is initially normal but may change as the animal ages. The inner adaxonal tongue is sometimes enlarged, but the relevance of this finding is unclear.

As expected, the axonal swellings are followed by waller-ian degeneration and are accompanied by strong activation of microglial cells and reactive astrogliosis, but no inflammation. Astrogliosis is more prominent in the corpus callo-sum, whereas reactive microglia are increased most in cerebellum. Compared to the subcortical white matter, glio-sis is less pronounced in the spinal cord. The reason for this regional heterogeneity is not known.

Taken together, CNP-mutant mice demonstrate more clearly than PLP mutants that two functions of oligodendrocytes, CNS myelin assembly and long-term axonal preservation, can be genetically uncoupled.

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