C. W. M. Adams

PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically‐defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of ‘normal’ white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter‐white matter distant from plaqueclose white matter‐edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base‐line of thin‐layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

ENZYME INACTIVITY OF MYELIN: HISTOCHEMICAL AND BIOCHEMICAL EVIDENCE

EARLIER work on the lipid and protein metabolism of grey and white matter of brain suggested that the myelin sheath may be in a metabolically dynamic state (SCHEINBERG and KOREY, 1962). Quantitative cytochemical studies of Ammon’s horn in the rabbit (LOWRY et af., 1954) first suggested that myelin may itself be enzyinically active. This view was based on finding more enzyme activity and more acid soluble phosphorus derivatives in the myelinated layer than would then be expected from the content of axons and neuroglia. More recently, it has been claimed that a wide range of oxidative and hydrolytic enzymes are present i n the myelin sheath of the peripheral nerve and in its neurokeratiri network (WOLFGRAM and ROSE, 1960; TEWARI and BOURNE, 1960a, b and c): some enzyme activity has also been reported in CNS myelin (TEWAIU and BOURNE, 1962n and h). TEWARI and BOURNE regard the tieurokeratin network as the locus of metabolic activity within the myelin sheath. If these histological and histochemical observations on neurokeratin and myelin enzymes can be substantiated, the myelin sheath would appear to be potentially metabolically active and could be regarded as a functional part of the cytoplasm of its formative cell-oligodendrocyte or Schwann cell~-and therefore, susceptible to injury by metabolic derangements. However, evidence has been accumulated from isotope studies to show that myelin lipids (DAVISON et al., 1959 a and 6; DAVISON and D o B B I N c ; , 1960) and proteins

THE OCCURRENCE OF ESTERIFIED CHOLESTEROL IN THE DEVELOPING NERVOUS SYSTEM

J ~ I E R I F I E D cholesterol, in relatively large amounts, has been found in the brain of developing chickens by MANDEL, BETH and STOLL (1949). The presence of these esters in the young animal and their apparent absence (SPFXRY, 1955) in the adult led us to postulate that esterified cholesterol may play an important role in the development of the central nervous system. This paper describes histochemical observations on the developing CNS of man and chickens, together with parallel chemical and chromatographic determinations of cholesterol and its esters. Evidence is presented to show that part of the ester cholesterol is located in the newly formed myelin sheath. The relevance of these histochemical and chemical observations to the process of myelinogenesis is also discussed.

PROTEINS, LIPID-PROTEIN DISSOCIATION AND PROTEINASE ACTIVITY IN WALLERIAN DEGENERATION

THE process of demyelination in Wallerian degeneration may be pertinent to the aetiology of the human dernyelinating diseases, since the study of this process may indicate where the causes of these diseases should be sought. As far as the myelin lipids are concerned, it has been shown that there are no chemical changes during the first week after nerve section, but thereafter the lipids are progressively degraded to cholesterol esters (JOHNSON, MCNABB and ROSSITER, 1949, 1950; ROSSITER, 1955). Likewise. the histochemical characteristics of the myelin lipids remain unaltered during the first week of demyelination (NOBAK and MONTAGNA, 1952; NOBAK and REILLY, 1956) but in the second week, cholesterol esters accumulate in the degenerating sheath as is shown by the positive Marchi reaction of the myelin break-down products at this stage (ADAMS, 1958, 1960). The early events in the first week of demyelination were attributed by ROSSITER and his colleagues to ‘physical’ disintegration of the sheath. However, although they showed that the myelin lipids were chemically unaffected in this preliminary phase no account was taken of changes in the myelin proteins or the bonding between lipid and protein in the sheath. It could be postulated that a protein constituent of myelin is chemically degraded during this early phase of dernyelination or, alternatively, that there is a disruption of lipid-protein bonds, such as those of CNS proteolipid (FOLCH and LEES, 1951) or the trypsin-resistant protein residue (TRPR)? lipid complex of the PNS (TUQAN and ADAMS, 1961). In support of the former hypothesis, it was shown by TUQAN and ADAMS that proteolytic enzymes cause in uitro ‘dernyelination’ apparently as a result of the liberation of lipid bound to a trypsin-digestible protein in the myelin of both CNS and PNS. The purpose of this investigationwas to determine by biochemical and histochemical means (a) whether there is any degradation of proteolipid protein and TRPR protein in degenerating myelin of the CNS and PNS, (b) whether there is disruption of the TRPR-lipid complex during demyelination and (c) whether proteolytic enzymes are present or become active in the degenerating nerve.

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.

HISTOCHEMISTRY OF MYELIN. XIII DIGESTION OF BASIC PROTEIN OUTSIDE ACUTE PLAQUES OF MULTIPLE SCLEROSIS

Abstract— Plaques of multiple sclerosis from a patient with a short clinical history were investigated by qualitative and quantitative histochemical methods. One of three plaques examined showed perivenous lymphocytic infiltration; this plaque was regarded as a particularly early acute lesion. In this plaque a relatively wide zone of diminished staining for basic protein extended outwards around the edge of the lesion. A narrower and irregular zone of diminished cerebroside staining was also seen around the plaque. Staining for phosphoglycerides and cholesterol was relatively normal up to the edge of the lesion; no zone of reduced staining for these lipids was seen outside the plaque. Proteolytic activity was increased throughout the lesion (pH 3.5 > 7.4). The two less acute plaques showed no obvious loss of basic protein and cerebroside outside the plaque. Addendum: Two further acute plaques of multiple sclerosis obtained recently, showed a similar loss of basic protein outside of lesion.

HISTOCHEMISTRY OF MYELIN. III. PERIPHERAL NERVE CATHEPSIN

Some of the properties of peripheral nerve cathepsin were investigated by histochemical and biochemical means. This cathepsin appears to be a constituent of the myelin sheath but its presence in the axon as well cannot be excluded. The enzyme is thermostable, sulphydryl-dependant and resistant to chloroform, methanol and acetone. It is mainly associated with the lipid and proteoplipid fractions of the nerve and this distribution supports the view that the enzyme is a constituent of the myclin sheath itself.

The relationship between enzyme activity and neuroglia in the prodromal and demyelinating stages of cyanide encephalopathy in the rat

In a previous communication (Ibrahim and Adams, 1963), we suggested that the increased enzymatic activity at the edge of the old yet active plaque of multiple sclerosis was due to the increase in oligodendrocyte population in this zone. Our observations on the multiple sclerosis plaque gave no indication whether this increase in neuroglial population and enzyme activity was a primary or secondary event in the demyelinating lesion. For this reason we have studied the neuroglial and enzyme changes in the prodromal stage of the experimental demyelinating disease produced by the injection of cyanide into the rat.

HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower‐moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower‐moving major protein, the ‘J’ band. The ‘J’ band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that ‘J’ band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion ‘J’ band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin‐sensitive, the basic protein being relatively more and the ‘J’ band protein relatively less susceptible.