H. Frey

ACID PROTEINASE ACTIVITY OF WHITE MATTER AND PLAQUES IN MULTIPLE SCLEROSIS

Recent evidence suggests that lysosomal acid proteinase of neuronal and glial cells are involved in the pathogenesis of demyelination (Hallpike et al. 1969). These lysosomal enzymes of central nervous system (CNS) may, however, also originate from hematogenous cells (Lampert 1967). Acid proteinase may liberate the basic protein from the myclin sheath (Hallpike e t al. 1969). Furthermore, it is tempting to suggest that the liberated encephalitogenic protein could induce immunization in the extracerebral round cells and lymph modes (Seitelberger 1967). However, typical multiple sclerosis (MS) lesions contain, in the border-zone of plaques, only a small number of these inflammatory cells (Ptr ier & Gregoire 1965, Suzuki et al. 1969). Therefore, at least in later stages of the disease, also cells originating from brain tissue may play a role in the autophagocytotic reactions. Enzymic changes in M S brains occur mainly in the border-zone of active plaques (Adams 1965). As acid proteinase may play a major role in the provocation of autoimmunity of MS (vide supra) and as the enzymic changes cannot be quantitated by histochemical means, the present work therefore gives data on the topographic changes in total specific activities of acid proteinase of MS brains.

Myelin basic protein antibodies in the serum and CSF of multiple sclerosis and subacute sclerosing panencephalitis patients.

A solid‐phase radioimmunoassay was developed for the detection of myelin basic protein antibodies of immunoglobulin G (IgG) class. Purified basic protein of myelin (MBP) was adsorbed onto polystyrene beads, followed by incubation in dilutions of serum or cerebrospinal fluid (CSF). 125I‐labelled anti‐human IgG was used to quantify antibodies bound to the solid‐phase. The assay was optimized in tests with rabbit antibodies to MBP and with 125I‐labelled anti‐rabbit IgG.

White Matter Proteins in Multiple Sclerosis

Abstract: The SDS‐soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non‐myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high‐molecular‐weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non‐myelin protein were also a consistent feature of the normal‐appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.

Measurement of glial fibrillary acidic protein (GFAP) and anti‐GFAP antibodies by solid‐phase radioimmunoassays

A solid‐phase radioimmunoassay was developed for the detection of glial fibrillary acidic protein (GFAP) and GFAP‐specific immunoglobulin G (IgG) antibodies. In antibody assays purified GFAP was adsorbed onto polystyrene beads, followed by incubation in dilutions of serum.

Critical evaluation of the postmortem factors influencing neurochemical analyses of brain autopsies

The effect of autolysis in both human and animal brain material was studied experimentally stored at different conditions. Four cell injury enzymes: acid proteinase, acid phosphatase, β‐glucuronidase and leucine aminopeptidase; four neurotransmitter synthesizing and metabolizing enzymes: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), glutamic acid decarboxylase (GAD), acetylcholinesterase (AChE) and the concentration of one neurotransmitter, gamma‐aminobutyric acid (GABA), were studied.

Pathogenesis of Multiple Sclerosis

In the present study, six multiple sclerosis (MS) brain white matter biopsies were analyzed both morphologically and chemically. The purpose of this study was to test the reliability of the earlier postmortem observations. Special attention was given to proteins which were analyzed in polyacrylamide disc gel electrophoresis and to acid proteinase. The results show that myelin basic protein (BP) was present in all biopsies, although advanced demyelination was seen in one case as based on morphology. This finding is controversial to earlier observations. In addition to this, some acid proteins were decreased or lost in two cases. The activity of acid proteinases was increased from two- to fivefold in four of six cases. This increase of the activity was seen also in the normal-appearing MS autopsy white matter which served as control. The source of this activity is discussed. On the basis of the present results it is likely that the role of the BP in the breakdown of myelin in MS and the role of acid proteinases has to be studied in more detail.

Myelin breakdown and basic protein.

Abstract The purpose of the present work was to study the mechanisms of myelin breakdown in experimental conditions and the fate of protein components in myelin, especially that of myelin basic protein which is known to be important in demyelinative disorders. The results showed that when myelin was incubated alone without any added enzymes there was a dissociation of basic protein from the myelin in acidic conditions but at neutral pH there was no change in basic protein. Whereas when myelin was incubated in the presence of acid proteinase-containing fraction there was profuse breakdown of basic protein and changes were also noted in the proteolipid protein. When separated myelin was incubated in the presence of neutral proteinase-containing fraction which was isolated from brain it was seen that it did not result in any breakdown fractions. When myelin was incubated in the presence of trypsin-containing fraction there was a complete loss of basic protein after 2 hr. These results suggest that the influence of acid proteinase is quite different from that of trypsin. This work also supports the theory that the breakdown of myelin BP is mediated through acid proteinases, while the action of neutral proteinase seems to be very slow, almost nonexistent.

Late-onset malignant astrocytoma in a case of multiple sclerosis

SummaryAn ununsual case of concurrent MS and anaplastic astrocytoma is presented.MS was diagnosed in a female patient at the age of 22 years. A left side thalamotomy was performed for relief of severe intention tremor at age 28 and at age 32 she received immunosuppressive therapy for 1 year. At the age of 36 after a severe exacerbation of her symptoms a left side fronto-temporal tumor was diagnosed and a subtotal neurosurgical extirpation was performed. Histopathologically, the tumor was an anaplastic astrocytoma, which was further substantiated by electron microscopy and establishment of a permanent cell line in vitro. The cultured tumor cells were negative for measles virus by immunofluorescence. The relationship between the reactive astrocytes in MS plaques and astrocytic neoplasia is discussed.

Esterases in developing CNS myelin

Abstract Changes in the car☐ylic acid esterase activities from crude myelin fractions and subfractions of myelin were followed during the development of guinea-pig brains. Animals from 15 days to 1 year of age were used. A rapid decrease of activity towards α-naphthyl acetate and β-naphthyl acetate was observed especially in the first myelin-like (M2) fraction. This decrease was somewhat more moderate in the second myelin-like (M2) fraction. On the other hand, α-naphthyl propionate and β-naphthyl laurate activities were quite different. Only minor changes could be demonstrated in the M2 fraction during myelination, and there were no significant alterations in the second myelin-like fraction during the same period. These results fit with earlier findings concerning changes in short- and long-chain fatty acid patterns of myelin during development.

Isoelectric focusing of myelin membrane proteins

Human myelin was isolated from the white matter of autopsy brains. Myelin proteins were characterized by isoelectric focusing in ultrathin slab gels in a pH range from 3.5 to 10 after solubilization with urea and Nonidet P 40. The protein profile in the acidic region (pH below 6.2) revealed at least twelve faint bands which comprised only a few percent of the total myelin proteins. Most of the myelin proteins were focused in the neutral range (pH 6.2–7.8) which showed two sharper and three broader major bands, the total number of bands in this region being about twenty. The basic pH range (pH above 7.8) contained about 30% of the proteins, and revealed a very intense band near the cathode with seven to nine weaker bands below pH 9.0. When the myelin was partially delipidated prior to solubilization, an additional broad “band” was observed at the area pH 8.0–8.5.