Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia

Abstract

Oligodendrocytes are critical for the development of the plasma membrane and cytoskeleton of the axon. In this paper, we show that fast axonal transport is also dependent on the oligodendrocyte. Using a mouse model of hereditary spastic paraplegia type 2 due to a null mutation of the myelin Plp gene, we find a progressive impairment in fast retrograde and anterograde transport. Increased levels of retrograde motor protein subunits are associated with accumulation of membranous organelles distal to nodal complexes. Using cell transplantation, we show categorically that the axonal phenotype is related to the presence of the overlying Plp null myelin. Our data demonstrate a novel role for oligodendrocytes in the local regulation of axonal function and have implications for the axonal loss associated with secondary progressive multiple sclerosis.

Figure 1.

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 axo–glial junction (arrow 2) is a highly specialized intercellular junction (shown in more detail in Fig. S1 D, available at http://www.jcb.org/cgi/content/full/jcb.200312012/DC1). (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. In this particular example only one axonal mitochondrion is evident (arrow), although in other instances several organelles may be present. Bar, 2 μm. (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 μm. (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 μm. (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 μm. (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 μm. (H) Another axon shows accumulation of dense bodies distal to the paranode (P). Bar, 1 μm.

Figure 2.

Absence of PLP/DM20 in oligodendrocytes does not impair the differentiation of axonal cytoskeleton and axolemma. (A) Axonal diameter frequency distributions from optic nerves of P60 wild-type and Plp null mice; the profiles are identical. (M ± SEM; n = 4). Inset: axonal densities in the optic nerve are also unaltered in the Plp null mice at P60. (M ± SEM; n = 4). (B) Levels of NF proteins in the triton-insoluble fraction from optic nerves of P60 Plp null mice are not different from wild-type littermates. (M ± SEM; n = 4 or 5). Similar results were found using the cytoskeletal- enriched fraction from brain and spinal cord (C). Representative immunoblots: NF-L recognizes the light chain NF, SMI-31 recognizes phosphorylated epitopes on the NF-H (h) and NF-M (m) polypeptides; RT-97 recognizes a different phosphorylated epitope on NF-H, and SMI-32 detects nonphosphorylated epitopes on NF-H. (D) Molecular markers for the node, ankyrin (red) and axolemma of the paranode, caspr-1 (green), and juxtaparanode, Kv1.1 (green) in P60 optic nerves from wild-type (WT) and Plp null (KO) mice. The localization appears similar in both genotypes. The Kv1.1 staining of the juxtaparanode is separated from the node by a gap, which represents the paranodal region. Bar, 5 μm.

Figure 3.

PLP/DM20-deficient oligodendrocytes induce a focal axonopathy when transplanted into the dorsal columns of shiverer mice. (A and B) Adjacent longitudinal resin sections of shiverer spinal cord transplanted with Plp null cells and immunostained for MBP (A) and PLP (B). The majority of myelin is in the dorsal columns. The transplanted myelin stains strongly for MBP (A), but is negative for PLP (B). Bar, 1 mm. When using this method of processing on resin sections, only the compact myelin of the transplant is immunostained. (C) shiverer axons are ensheathed, rather than myelinated, by oligodendrocyte processes that are PLP+/MBP−, and a cryosection immunostained for PLP (green) and MBP (red) shows two fibers at the junction between the host shiverer processes (shi) and the PLP−/MBP+ myelin produced by the transplanted Plp null cells (null). Bar, 20 μm. (D) Myelination by transplanted wild-type cells showing a node (N) and normal axonal appearance. Bar, 2 μm. (E) Part of the wild-type myelin sheath with major dense (M, arrow) and intraperiod (I, arrow) lines. Bar, 50 nm. (F) Myelination by transplanted Plp null cells showing an axon containing numerous membranous organelles adjacent to a paranode (P). Bar, 2 μm. (G) Part of the PLP-deficient myelin sheath showing widening of the intraperiod line. Bar, 50 nm.

Figure 4.

Absence of PLP/DM20 is associated with impaired fast retrograde and anterograde axonal transport. (A) Schematic of the optic pathways showing the eye, optic nerve (ON), chiasm (C), optic tract (OT), and superior colliculus (SC). The distal optic tract continues as the brachium of the superior colliculus (not depicted) to terminate in the optic nerve layer of the colliculus. In rodents, >90% of axons in the optic nerve decussate at the chiasm and terminate in the contralateral superior colliculus. The RGCs are the cell bodies of axons that terminate in the colliculus. (B) Area of retinal whole mount from wild-type (WT) and Plp null (null) littermates aged 4 mo, 12 h after injection of FITC-conjugated CTB into the superior colliculus; images were collected and processed identically. The number of labeled RGCs and the intensity of fluorescence are much greater in the wild type. Bar, 50 μm. (C) Accumulation of retrogradely transported CTB-FITC 12 h after injection into the superior colliculus in wild-type and Plp null littermates of denoted ages. The signal in the RGCs is expressed as fluorescence area (μm²/mm²) of retina. (M ± SEM; n = 4 or 5). The difference between the two genotypes is significant at all ages except 1 mo. (D) Frequency distribution of RGC body area as defined by immunostaining for β-tubulin III in wild-type and Plp null littermates at P60. (M ± SEM; n = 2). There is no difference between the two genotypes. (E) Longitudinal sections of optic nerves from wild-type (WT) and Plp null mice at 4 mo of age, 48 h after injection of FITC-conjugated CTB into the superior colliculus. Only an occasional focus of fluorescent labeling is observed in the wild type, whereas numerous CTB-filled axonal swellings are evident along the Plp null nerve. The direction of retrograde transport is indicated by the arrow. Bar, 200 μm. (F) Enlargement of single swelling from E, showing a “tail” of tracer in the axon distal to the swelling. Bar, 10 μm. (G) Section from the nerve shown in E, immunostained for caspr-1 (red) and NFs (blue). The FITC-conjugated CTB has accumulated distal to the nodal complex. The node (arrow) is bounded by the caspr-1–stained paranodes. The caspr-1 staining above and below the swelling belongs to other axons. Note the punctate staining of the CTB, suggesting its presence in membranous components such as endosomes. Bar, 5 μm. (H) Accumulation of fast anterogradely transported protein in the superior colliculi of wild-type and Plp null mice at ages 2, 4, and 18 mo. The radiolabeled protein in the colliculi is expressed as a percentage of the total TCA-precipitated labeled material in the optic system. (M ± SEM; n = 5–7). The differences at 4 and 18 mo are significant.

Figure 5.

Absence of PLP/DM20 is associated with elevated levels of retrograde motor proteins in affected regions. (A–F) Steady-state levels of dynein intermediate chain, dynactin p50, kinesin heavy chain, Lis1, and Nudel in triton-extracted fractions of optic nerve (A and B), spinal cord (C and D), and cerebral cortex (E and F) of Plp null mice at P60. Bar charts (A, C, and E) show the percent change from wild-type littermates (M ± SEM; n = 3–7); panels (B, D, and F) show representative blots. The protein loading for dynein in the wild-type spinal cord is threefold greater than the null (3:1). In the optic nerve, all values except kinesin are significantly elevated in the Plp null mice. In the spinal cord dynein, dynactin and Lis1 are increased, whereas in the cerebral cortex there is no difference in any parameter (G). Representative RT-PCR of dynein intermediate chain genes 1 (Dnci1) and 2 (Dnci2) from retinae of P60 wild-type and Plp null mice. Two alternatively spliced Dnci1 transcripts (1a and 1c) are present. Cyclophilin (Cyclo) acts as an internal control. There is no difference in signals between the two genotypes.

Figure 6.

Late-onset degeneration in the rostral cervical fasciculus gracilis of mice aged 18 mo. (A) Wild-type (WT) mouse. (B) Plp null (Plp−/y) mouse showing axonal degeneration. (C) Plp heterozygous (Plp^+/−) mouse showing a pattern of degeneration very similar to that occurring in the null mouse. (D) Plp null mouse carrying the Wlds mutation (Wlds) that retards Wallerian degeneration after axonal transection. The pattern of axonal degeneration is very similar to that in the unmodified Plp null mouse, indicating the Wlds mutation provides no obvious protection. Bar, 50 μm. Insets: the fasciculus gracilis at higher power with the normal appearance in the wild-type mouse, and changes typical of axonal degeneration (arrow) in the other three genotypes. Bar, 10 μm.