Catherine Mezei

Temporal changes in PO and MBP gene expression after crush-injury of the adult peripheral nerve

The crush-injured sciatic nerve provides a model to study Schwann cell regulation of myelin gene expression during the process of demyelination and remyelination. In order to investigate the possible transcriptional regulation of myelin gene expression, the quantity, quality and translational efficiency of PO (the major myelin glycoprotein) and MBP (the myelin basic proteins) coding messages were investigated as a function of time following crush-injury of the adult rat sciatic nerve. Northern blot analysis indicated that the size of the PO and MBP transcripts remain unchanged in the distal segments of crushed sciatic nerves at 1, 2, 4, 7, 10, 14 and 21 days after crush-injury. Dot-blot analysis showed a sharp drop in levels of PO and MBP coding transcripts 1 day after crush-injury with the lowest steady-state levels at 4-7 days. Message levels were found to increase after 7 days, the highest increase in levels of message was found to be between 10 and 14 days. The highest steady-state level of both transcripts was observed at 21 days. In vitro translation and immunoprecipitation of PO-translated products from various stages of crush-injury also indicated this trend. The pattern of gene expression of PO- and MBP-coding transcripts parallel each other and follow the pattern of demyelination and remyelination. The results are also consistent with our previous interpretation which suggests that PO and MBP gene expression is regulated at the level of transcription and that these two genes might be coordinately expressed. Western blot analysis of PO protein from these stages revealed a similar decrease and then increase in the levels of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression in the presence or absence of myelin assembly

Regulation of myelin protein gene expression in the presence and absence of myelin assembly can be assessed using crushed or permanently transected adult sciatic nerves of rats. The P0 glycoprotein and the myelin basic protein (MBP) are the major myelin-specific proteins of the peripheral nervous system. The steady-state level of P0 and MBP messenger RNA was determined by dot-blot analysis of poly(A)+ RNA from crushed and transected nerves of rats at 35 days post operation. The rat P0-specific cDNA clone, pSN63c, and mouse MBP-specific cDNA clone, pHF43, were used as probes. The level and quality of the poly(A)+ RNA was assessed by in vitro translation and immunoprecipitation of the translation products with anti-chick P0 antibody. Comparison of the steady-state level of P0 and MBP transcripts and the level of anti-P0 immunoprecipitated translation products from RNA extracts of permanently transected, crushed, adult control and 21-day-old control rat nerves indicated that the level of P0 and MBP messages was significantly reduced in the permanently transected model, whereas it was restored to normal in the crushed sciatic nerve 35 days post injury. These results suggest that regulation of P0 and MBP gene expression most likely occurs at the transcriptional or post-transcriptional level in the two models of peripheral neuropathies. Northern blot analysis indicated the absence of differential splicing of the message in crushed or transected nerves. The experiments also indicate that these two important gene products required for myelin synthesis and assembly seem to be co-regulated. However, the data do not rule out the possibility that regulation of gene expression may also occur at the level of translation or post-translational processing.

Myelin-associated glycoprotein gene expression in the presence and absence of Schwann cell-axonal contact

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.

Solid‐Phase Immunoassay of PO Glycoprotein of Peripheral Nerve Myelin

Abstract: 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 γ‐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.

Effect of Wallerian Degeneration on Histamine Concentration of the Peripheral Nerve

One sciatic nerve of a White Leghorn hen was severed and the distal portion was allowed to undergo Wallerian degeneration. The change in histamine and DNA concentration and mast cell number was measured at different times following nerve sectioning in the proximal regenerating, distal degenerating, and intact, contralateral nerves. The experimental results revealed a significant accumulation of histamine in the proximal desheathed segment and in the contralateral “functional nerve,” whereas the biogenic amine in the distal desheathed nerve significantly decreased. The pattern of change of histamine in the distal and proximal nerve sheaths was different: it dropped at 2 h and then rose in the later stages of Wallerian degeneration. In the distal desheathed nerves and in both the proximal and distal nerve sheaths DNA increased significantly by 14 days. The number of mast cells appeared to be highest in the 14‐day distal nerve and in the 7‐day proximal nerve sheaths. These results support a dual localization of histamine in the peripheral nerve, and are consistent with the interpretation that the amine has either some role in neurotransmission or in the process of growth and regeneration.

Cholesterol esters and hydrolytic cholesterol esterase during Wallerian degeneration.

To study the involvement of cholesterol esters in myelination and demyelination, we determined the concentration of free cholesterol and cholesterol esters and the activity of hydrolytic cholesterol esterase (sterol ester hydrolase; EC 3.1.1.13) in hen sciatic nerve during Wallerian degeneration. A progressive increase in the ratio of cholesterol ester to free cholesterol was observed in the degenerating nerve at 8, 16 and 32 days after nerve section. Hydrolytic cholesterol esterase activity decreased progressively in the degenerating nerves at the same time.

Levels of Proteolipid Protein mRNAs in Peripheral Nerve Are Not Under Stringent Axonal Control

Abstract: 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 ∼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 chick sciatic nerve myelin: purification and partial characterization.

Abstract: 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 electro‐phoresis (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.

Ontogenesis of histamine in the chick nervous system.

Tissues from the central and peripheral nervous systems of the chick were analyzed for concentration of histamine (Hm) during development. Of the three CNS organs examined, cerebral hemispheres had the highest Hm content. Expressed on the bases of wet weight, protein, and DNA concentrations, sciatic nerve and the pineal gland had the highest levels of this biogenic amine of the five tissues investigated. The concentration of Hm was higher in the cerebellum, cerebral hemispheres, and thalamus of adult animals than in the 15 to 17-day-old embryos. The level of Hm rose markedly in the sciatic nerve and pineal gland after the 15th day of embryonic development. These data might indicate a possible involvement of Hm in controlling the course of maturation of certain organs in the nervous system.

A 42 K protein of chick sciatic nerve is immunologically related to PO protein of peripheral nerve myelin

The major protein (PO) in PNS myelin is an integral membrane glycoprotein with a molecular weight of about 30 K. The level of PO protein in the developing sciatic nerve of the chicken was monitored by a solid-phase immunoassay and densitometry of Coomassie blue stained polyacrylamide gels. The most rapid rate of accumulation of PO protein occurred after 16 days of embryonic development. In addition to the 30 K PO protein, a number of higher molecular weight proteins could be distinctly detected by immunoblotting. Amongst these high molecular weight proteins, a species with an apparent molecular weight of 42 K was specifically immunostained with epitope-selected polyclonal antibodies against PO protein. This 42 K protein could be first detected after 16 days of embryonic development and increased rapidly following the pattern of myelination in the sciatic nerve. The enzyme endoglycosidase F, which specifically removes N-asparagine linked high mannose and complex carbohydrates from glycoproteins, converted the PO and 42 K proteins to lower molecular weight forms, which could be specifically immunostained by epitope selected polyclonal antibodies to the PO protein. Subcellular fractionation of the 17-day embryonic nerve demonstrated that the 42 K protein was enriched in myelin and microsomal subfractions relative to the total homogenate. These results indicate that the 42 K immuno-crossreactive protein might be chemically and functionally related to the PO protein of the PNS myelin.