P.J. Riekkinen

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.

Brain Glutamic Acid Decarboxylase Activity in Parkinson’s Disease

The activity of glutamic acid decarboxylase (GAD), the enzyme involved in formation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), was studied in autopsy brain samples from six parkinsonian patients and 13 controls. There was relatively good postmortem stability of GAD in deep-frozen brain samples over a 3-week period. The activity of GAD was significantly reduced in brain samples of patients with Parkinson’s disease, being about 50% of that in controls. Moreover, levodopa treatment showed a tendency to increase the activity of GAD. The results suggest the involvement of GABA neurons in Parkinson’s disease.

Studies on the pathogenesis of multiple sclerosis. 2',3'-Cyclic nucleotide 3-phosphohydrolase as marker of demyelination and correlation of findings with lysosomal changes.

Abstract 2′,3′-Cyclic nucleotide 3-phosphohydrolase, an enzyme proven in earlier studies to be associated with myelin, was used as indicator of demyelination in autopsy specimens of brain in 8 cases of multiple sclerosis and 2 of subacute sclerosing panencephalitis. Our results revealed that areas which showed demyelination on light microscopy showed also decreased phosphohydrolase values. The activity of phosphohydrolase increased in areas immediately outside plaques. These results compared well with cerebroside and other lipid data. However it was found that there was some discrepancy between lipid amounts and phosphohydrolase activity. Some areas where phosphohydrolase values were 3–4 times lower than in corresponding control specimens showed only half of the normal cerebroside content. The so-called normal white matter outside plaques did not show any constant decrease of phosphohydrolase activity but our material is too limited for any conclusion to be drawn. Our second major finding in this study was an increased response of lysosomes in areas where phosphohydrolase or lipid data did not reveal significant pathological changes. These results suggest that before demyelination begins there is a cellular response, possibly in glial cells, and demyelination seems to be only one of the consequences of this response.

A critical evaluation of myelin purification. Non-specific esterase activity associated with central nerve myelin preparations

Abstract The isolation of myelin from bovine bovine brain tissue has been investigated in detail by analysis of contaminating cell structures at different stages of the purification procedure. The effect of a hypo-osmotic shock treatment on the purity of myelin fractions has been studied. Contamination in myelin preparations has been evaluated using a combination of techniques. The problems of finding suitable criteria for assessing myelin purity are discussed. Repeated density gradient centrifugation of myelin preparations was found to be essential in reducing levels of contaminating cell structures to a minimum. Homogenisation of myelin in water during the isolation procedure was also important in achieving negligible impurity in final preparations. High purification factors have been achieved in final myelin samples for succinate dehydrogenase, lactate dehydrogenase, acid phosphatase and acid proteinase. A homogenisation and centrifugation technique has been used to separate microsomes from a crude myelin fraction. Non-specific esterase activity can be demonstrated in final highly purified myelin samples. The enzyme activity is considered to be directly associated with the myelin lamellae.

Further studies on neutral proteinase activity of CNS myelin.

Abstract CNS myelin was prepared by six different gradient systems. The purity of the preparations was evaluated by electron microscopy and chemical markers. Electron-microscopic analysis of crude myelin preparations revealed contamination by axonal contents, mitochondria and lysosomes, while final preparations were characterized by small myelin fragments with little or no contamination. Chemical analysis revealed an approximately 500–1000-fold decrease in soluble, mitochondrial and lysosomal markers. The final preparations revealed a typical myelin structure and an absence of significant contamination by DNA. 7–14% of the neutral proteinase activity of the crude brain homogenate was found in purified myelin. This may favour the possibility that a part of the neutral proteinase is intimately linked to the myelin. The possible function of neutral proteinase in the formation of myelin buds during demyelination is discussed.

BRAIN ACETYLCHOLINESTERASE IN PARKINSON'S DISEASE

The activity of acetylcholinesterase was studied in various autopsy brain samples of nine parkinsonian patients and eight control subjects without extrapyramidal disorders. Putamen and caudate nucleus showed the highest activities of acetylcholinesterase when different brain areas were compared. Acetylcholinesterase values expressed per wet weight or protein were somewhat lower in the extrapyramidal brain regions of parkinsonian patients than in those of controls. When values were calculated per desoxyribonucleic acid (DNA) it was found that extrapyramidal brain regions, especially substantia nigra, showed higher activities in parkinsonian material than in controls. This difference is due to the decrease of DNA which in the substantia nigra mainly reflects the loss of dopaminergic substantia nigra neurons. Furthermore, analyses of dopamine from the same tissue samples showed many times increased acetylcholinesterase‐dopamine ratio in parkinsonian brain than in controls. Levodopa alone or combined with a decarboxylase inhibitor did not have any significant effect on the activity of acetylcholinesterase. It is concluded that the cholinergic mechanisms in the extrapyramidal system of parkinsonian brain are not so severely affected as the dopaminergic ones leading to relative and functional cholinergic dominance.

Guillan-Barré Syndrome

A 66-years-old man suffering from Guillan-Barre syndrome (GBS) died 11 days after the onset of the disease and was autopsied 12 h later. The histopathological preparations showed oedema and demyelinat

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.

Neurochemical and morphological studies on demyelination in multiple sclerosis with special reference to etiological aspects

SummaryLight microscopic studies were used as control for neurochemical studies and these showed that some micro plaques could be found also in areas which were normal on visual inspection. Also foreign cell infiltrates were found outside any clear plaque material. The number of these cells did not correlate with other findings like lipid or enzyme chemistry. In electronmicroscopic studies astrocytes demonstrated most lysosomes and phagocytosis of myelin. This increased lysosomal reaction was demonstrated also in biochemical analyses performed on MS biopsy specimens. Occasional nuclear changes like inclusion bodies and protrusion of inner nuclear membrane were observed suggesting some exogenous, possibly viral factor as an origin of the disease. Neurochemical studies showed that demyelination as a sense of decrease in myelin lipid and phosphohydrolase activity is only a later phenomenon and increased lysosomal activities possibly originating from various glial cells are found before demyelination. However myelin-like material found inside lysosomes suggests the early moderate demyelination before classical plaques can be found. Acid hydrolases were mostly increased at areas around plaques and encephalitogenic protein was not demonstrable at plaque areas. Our combined study suggests early changes of glial cells as a basic mechanism of the disease. However, the clarification of the basic course of these changes remains to be seen although nuclear changes may suggest a slow viral infection.ZusammenfassungLichtmikroskopische Untersuchungen als Kontrolle neurochemischer Studien zeigten, daß einige Mikroplaques auch in Bezirken zu finden waren, die bei visueller Betrachtung normal erschienen. Weiterhin fanden sich Infiltrate fremder Zellen auch außerhalb des eigentlichen Plaquematerials. Die Zahl dieser Zellen korrelierte jedoch nicht mit anderen Befunden wie Lipid- und Enzymchemie. Elektronenmikroskopisch wiesen Astrocyten die meisten Lysosomen und die stärkste Myelinphagocytose auf. Diese verstärkte lysosomale Reaktion ließ sich auch an Hand der an MS-Biopsiematerial durchgeführten biochemischen Analysen nachweisen. Gelegentlich wurden auch nukleare Veränderungen wie Einschlußkörperchen und eine Protrusion der inneren Kernmembran beobachtet, die aufeinen exogenen, möglicherweise viralen Faktor als Ursache der Erkrankung hinweisen. Neurochemische Analysen zeigten, daß die Erkrankung im Sinne einer Abnahme der Myelinlipide und der Phosphohydrolyseaktivität erst später in Erscheinung tritt, während vermehrte lysosomale Aktivitäten, die von verschiedenartigen Gliazellen ausgehen, vor der Entmarkung zu finden sind. Myelin-ähnliches Material in den Lysosomen läßt jedoch vermuten, daß eine mäßige Entmarkung der Ausbildung klassischer Plaques vorausgeht. Saure Hydrolysen waren vornehmlich in der Umgebung der Plaques vermehrt, encephalitogenes Protein war im Bereich der Plaques nicht nachzuweisen. Unsere kombinierte Studie läßt vermuten, daß frühe Veränderungen der Gliazellen einen grundlegenden Mechanismus dieser Krankheit darstellen. Die Klärung des eigentlichen Verlaufes dieser Veränderungen steht jedoch noch aus, wenngleich nukleare Veränderungen auf eine langsame Virusinfektion hinweisen.

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.