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The distal segments of the crush-injured and permanently transected sciatic nerve provide models to study Schwann cell activity in the presence and absence of Schwann cell-axonal contact, respectively. We examined the quantity and quality of transcript coding for the myelin-associated glycoprotein (MAG) over a 3-week period following crush injury and at 35 days after transection to investigate possible regulation of this gene during nerve injury and subsequent repair. Northern blot and slot blot analysis indicated a sharp decrease in levels of MAG mRNA 2 days after crush injury which was followed by a progressive increase in levels of message between 7 and 21 days after injury. Western blot analysis showed that levels of MAG protein decreased substantially 7 days after crush injury, which returned to 70% of the adult value by 21 days after injury. MAG mRNA and protein were undetectable by Northern and Western analysis, respectively, in the distal segment of the sciatic nerve 35 days after permanent transection. This infers distinct down-regulation of MAG gene expression after permanent transection of a peripheral nerve. These comparative studies of MAG transcripts and encoded protein may indicate regulation of MAG gene expression at the level of transcription, and possibly at the level of post-transcription in these experimental models of peripheral neuropathies.

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were 1) down-regulated to basal levels (P0 and MBP mRNAs), 2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), 3) upregulated (mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase and beta-actin), or 4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons.

Abstract. The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-l-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.T HE myelin sheath of the peripheral nervous system (PNS) ~ is a vastly extended and modified plasma membrane of the Schwann cell (Geren, 1954). This membrane is wrapped around the nerve axon in a spiral fashion to form a tightly compacted, multilamellar structure. During development, Schwann cells accumulate along axons of growing peripheral nerves and proliferate until they completely engulf the axons (Speidel, 1964). Myelinogenesis is dependent on appropriate interactions of Schwann cells, axons, and extracellular matrix (Bunge, 1987). Apart from certain lipid precursors that may be obtained partially from the circulation or through axoplasmic transport from the neurons, the general consensus is that almost all myelin synthesis and assembly is carried out within the Schwann cell (Norton and Cammer, 1984). However, it is the nature of the axon that determines whether or not a given cell will produce 1. Abbreviations usedin thispaper: apo A-I, apolipoprotein A-l; HDL, high density lipoprotein; LDL, low density lipoprotein; PNS, peripheral nervous system; S-2, supernatant 2; SO, supernatant of osmotically shocked myelin; VLDL, very low density lipoprotein. myelin (Weinberg and Spencer, 1976;Br...

The proteolipid protein (PLP) is the major protein in the myelin sheath of the CNS. It was recently reported that PLP coding transcripts are also found in the PNS, although the protein was not detectable in peripheral nerve myelin. In the present investigation, levels of mRNA for PLP in sciatic nerve were studied during development and following transection and crush injury. Results were compared to those for P0, the major PNS myelin protein, and the myelin-associated glycoprotein (MAG). PLP transcript levels were very low at 21 days in sciatic nerve and remained unchanged in the adult sciatic nerve. This contrasts markedly with P0 and MAG mRNAs, which are expressed at high levels during development and decrease in content significantly by adulthood. The level of PLP messages was reduced approximately 40% in the quiescent Schwann cells in the distal segment of the sciatic nerve at 21 days after permanent transection, yet P0 mRNA levels were very low, and MAG mRNAs were undetectable in this tissue. The distal segment of the crush-injured sciatic nerve is characterized by transient demyelination followed by rapid myelination. PLP mRNA levels remained comparatively unaffected in the 3-week period following crush injury. RNase protection experiments using two antisense riboprobes confirmed that levels of PLP-derived protected fragments, corresponding to PLP and DM-20 messages, remained unchanged in the developing and adult sciatic nerve. These results indicate that myelin-specific P0 and MAG genes are tightly controlled at the level of transcription through Schwann cell-axonal interactions, whereas PLP transcription in the peripheral nerve remains nearly dissociated from axonal influences.

The PO protein of the myelin of chick sciatic nerve was isolated and purified by propanoic acid extraction of peripheral nervous system (PNS) myelin, delipidation, Sepharose CL-6B chromatography in the presence of sodium dodecyl sulfate (SDS), and preparative SDS-polyacrylamide gel electrophoresis (PAGE). Approximately 15% of the PO protein in the sciatic nerve myelin was recovered in a homogeneous state. The purified protein monomer has an apparent molecular weight of 32.1K as determined by gel electrophoresis. The PO protein undergoes extensive aggregation during exhaustive dialysis and freeze-drying and yields stable dimers, trimers, and tetramers. The aggregation of the PO protein after freeze-drying is independent of the presence of a reducing agent (2-mercaptoethanol) in the solubilizing medium. The PO protein is a glycoprotein. The amino acid composition of the chick PO protein indicates a definite species difference when compared with mammalian PO proteins although the NH2-terminal isoleucine residue seems to have been retained during evolution.

To explore the immunological properties of PO protein, antibodies were elicited in rabbits against the purified chick PO protein. Peripheral nervous system protein was fractionated on sodium dodecyl sulfate-polyacrylamide slab gels and then transferred electrophoretically ("blotted") onto nitrocellulose sheets. The PO protein was detected by its capacity to bind its specific antibody present in the rabbit serum. The PO-specific antibody complex was then exposed to goat anti-rabbit immunoglobulin G (IgG) coupled to peroxidase or labeled with 125I. The resulting PO antigen-antibody "sandwich" was visualized and quantitated by densitometry of the colored peroxidase reaction product or by autoradiography and gamma-radiation counting of the 125I-IgG complex. The methods permitted quantitation of the PO protein in various nerve extracts. The limit of detection of the PO antigen was about 1 ng of protein. The antibody was specific for the PO glycoprotein in the peripheral nerve extracts. The PO proteins from various species, including human, were also detected by the antibody to chick PO protein. Preliminary experiments indicate the solid-phase immunoassay is a useful method for monitoring PO protein levels in small quantities of tissue extracts under various physiological and pathological conditions.