Hilde E. Hirsch

ACID PHOSPHATASE LOCALIZATION IN INDIVIDUAL NEURONS BY A QUANTITATIVE HISTOCHEMICAL METHOD

—By an adaptation of the fluorometric method of Campbell and Moss (1961), the activity of α‐naphthyl acid phosphatase was measured in individual neurons of monkey and human spinal cord and found to be many times higher in nerve cell bodies than in the surrounding neuropil. It was also measured in cerebellar cortex and found most concentrated in the granular (neuronal) layer. As this distribution is distinctive and paralleled by two other acid hydrolases, β‐galactosidase and β‐glucuronidase, it is considered to offer additional support for the lysosomal concept in nervous tissue and to indicate that nerve cell perikarya are much richer in lysosomes than are axons, dendrites or glial cells.

A THIOL PROTEINASE HIGHLY ELEVATED IN AND AROUND THE PLAQUES OF MULTIPLE SCLEROSIS.: SOME BIOCHEMICAL PARAMETERS OF PLAQUE ACTIVITY AND PROGRESSION

A thiol dependent proteolytic enzyme (tentatively identified as carboxypeptidase B) which liberates phenylalanine from CBZ‐glutamyl‐phenylalanine at pH 5.3 was shown, by a sensitive micromethod, to be greatly increased in activity in and around MS plaques. These increases exceeded those of other hydrolases previously measured. Plaque tissue, on the basis of lipid‐free dry weight, is up to 3‐fold richer in this enzyme than control white matter, and most samples of apparently uninvolved MS white matter already show elevated activities. The enzyme is highly dependent on the presence of dithiothreitol. It is unaffected by diisopropyl fluorophosphate and pepstatin, but inhibited by iodoacetate and leupeptin. Macrophages or lymphocytic infiltrations in the tissue do not appear to be the main source of the enzyme.

LOCALIZATION OF ARYLSULPHATASE IN NEURONS

Abstract— Arylsulphatase activity, with 4‐methylumbelliferone sulphate as substrate, was measured by a quantitative histochemical method in individual anterior horn nerve cell bodies and adjacent neuropil of man and monkey; and in molecular and granular layers and subjacent white matter of cerebellum of monkey, rat and guinea pig. The activity was much higher in neuronal perikarya than in neuropil, and higher in the granular layer of cerebellum than in the molecular or white matter, thus resembling the distinctive distribution, reported in monkey, of three other lysosomal enzymes, β‐galactosidase, β‐glucuronidase and α‐naphthyl acid phosphatase. One exception was encountered: the white matter of guinea pig cerebellum had more arysulphatase activity than the granular layer. For comparison, other lysosomal enzymes also were measured in rat and guinea pig cerebellum; in these species, α‐naphthyl acid phosphatase distribution was found to differ from that of β‐galactosidase and arysulphatase, and from the pattern common to four lysosomal enzymes in the monkey.

Acid proteinases and other acid hydrolases in experimental allergic encephalomyelitis: pinpointing the source.

—In Lewis rats with experimental allergic encephalomyelitis (EAE), quantitative histochemical techniques were used to measure the activities of several hydrolytic enzymes, especially acid proteinases, and their levels were correlated with the concentrations of DNA and total lipids. Only those samples which contained lesions (visualized directly or in stained nearby sections) had elevated acid hydrolase activities. A lipase‐esterase hydrolysing a fluorogenic substrate at pH 5.0 was more active in lesions; activity on the same substrate at pH 7.2 was not elevated. β‐Glucuronidase rather than acid proteinase or cathepsin D was the enzyme showing the greatest differences. In a monkey with EAE, studied for comparison, a massive inflammatory lesion appeared to leak β‐glucuronidase to a distance of about 2.5 mm beyond its margin.

The quantitative histochemistry of acid proteinase in the nervous system: localization in neurons.

A new, fluorometric method was used to assay acid proteinase in tissue samples at great sensitivity. Digestion of hemoglobin at pH 3.8 (in brain mostly due to cathepsin D) was measured in individual nerve cell bodies and neuropil from the anterior horn of the spinal cord, and in the molecular and granular layers and while matter of cerebellum, in man and in monkey. Anterior horn cell perikarya were about 25 times more active than neuropil, and the granular layer of cerebellum had about three times the activity of the molecular layer. This predominantly neuronal localization resembles the distribution of other lysosomal hydrolases. The high capacity for protein breakdown found in neurons is in accord with their known high rates of protein synthesis and turnover.

Fibrinolytic Activity of Plaques and White Matter in Multiple Sclerosis

Recent work has implicated plasminogen activator released from macrophages as a possible mediator of the demyelinating process in experimental allergic encephalomyelitis and multiple sclerosis (MS). We have studied the capacity of white matter and plaques from MS patients to break down fibrin clots, using a histochemical technique. Fibrinolytic activity was localized exclusively to areas around blood vessels and capillaries in both patients and controls. While there was marked variation between individuals, the unaffected white matter from MS patients was, on the average, not more active than that of controls, but plaques tended to show more numerous foci of lysis, often also more intense, than adjacent white matter; there was no correlation with disease activity or age of the plaques as determined by histological criteria. The localization and degree of fibrinolysis observed were not related to the presence of lymphocytic infiltrates, gliosis, or macrophages. However, the findings do not exclude an involvement of fibrinolytic enzymes (although originating from vascular endothelium rather than macrophages) in the genesis of the MS plaque, which commonly starts around a small vein

Acid hydrolases and other enzymes in secondary demyelination. A quantitative histochemical study in the wobbler mouse.

Abstract— The hereditary motor neuron degeneration found in the wobbler (wr) mouse was studied as a model of secondary demyelination. Lysosomal enzymes (acid phosphatase, acid proteinase, β‐glucuronidase and β‐galactosidase) were found elevated about three‐fold in the white matter of the affected cervical spinal cord as compared with normal controls; but they were either not increased, or increased much less, in the anterior horn. Since gliosis and influx of phagocytic cells are minimal in this model, the high hydrolase levels are believed to arise primarily from (a) the accumulations of axonal dense bodies seen in involved areas, and (b) from indigenous cells engaged in breaking down the myelin fragments. Thus, secondary demyelination may, at least in this case, be initiated by enzymes of local origin.

Fluorometric oxidase assays: Pitfalls caused by action of ultraviolet light on lipids

A number of fluorometric assays of hydrogen peroxide-producing oxidases are based on the formation of highly fluorescent products from homovanillic acid or related compounds by horseradish peroxidase. We report the observation that under continuous uv illumination at the wavelengths used for excitation in these methods, brain or muscle homogenates produce fluorescence increases in the absence of any exogenous enzyme substrate; when uv light is excluded, such increases are negligible. Arachidonic and linolenic acids also produce this effect. For this reason, measurements of H2O2 based on this principle are valid only if this nonspecific effect has been excluded, and should preferably be carried out as end-point rather than continuous assays. It is believed that the effect of uv light on the reaction is due to formation of H2O2 and/or oxygen free radicals, and polyunsaturated lipids appear to be involved as intermediates. Thus, the homovanillic acid-horseradish peroxidase system may prove useful in investigations of the effect of uv on the production of oxygen free radicals and lipid peroxidation.

Acid lipase-esterase (4-methylumbelliferyl oleate hydrolase) of white matter localized in oligodendrocyte cell bodies.

Abstract— The localization of an acid lipase‐esterase which cleaves the fluorogenic substrate 4‐methyl‐umbelliferyl oleate at pH 5 was studied, because previous experiments has shown this activity to be reduced in the plaques of multiple sclerosis. In the human cerebellum, quantitative histochemical methods showed the activity to be relatively low in the molecular layer, compared to the granule cell layer; but the underlying white matter was the most active. In the human spinal cord, anterior horn cell bodies were richest in acid lipase, but white matter was, on a dry weight basis, as active as neuropil. Oligodendrocytes obtained in bulk from bovine white matter according to Poduslo & Norton (1972) had a specific activity up to 20 times greater than the crude myelin fraction. While it remains possible that in myelin the enzyme is in an inactive or inhibited state, the results indicate that the enzyme is localized in oligodendroglial cell bodies and suggest its use as a marker.

Glycerol Phosphate Dehydrogenase: Reduced Activity in Multiple Sclerosis Plaques Confirms Localization in Oligodendrocytes

Cytoplasmic glycerol phosphate dehydrogenase (snglycerol-3-phosphate:NADf 2-oxidoreductase, EC 1.1.1.8) (GPDH) in the nervous system has been found by immunocytochemical methods exclusively in oligodendrocytes (De Vellis et al., 1978; Leveille et al., 1979). We wish to report that chronic plaques of multiple sclerosis (MS), from which the oligodendrocytes have disappeared, show a dramatic loss in this enzyme. This loss parallels the decline in the plaques of the activity of acid lipase-esterase (4-methylumbellifery1 oleate hydrolase) (Hirsch et al., 1976; Hirsch and Parks, 1979) which, in white matter, appears to be localized in oligodendroglial cell bodies (Hirsch et al., 1977). The results are consistent with a predominantly, but perhaps not exclusively, oligodendroglial localization of this enzyme.