Globoid cell leukodystrophy (Krabbe disease) in a Merino sheep

Abstract

We describe the clinicopathologic features of an ovine case of Krabbe disease (globoid cell leukodystrophy). Brain lesions, sometimes bilaterally distributed, were present in the cerebellar peduncles, cerebellar folia white matter, medulla, pons, and spinal cord and characterized by marked myelin loss and numerous large macrophages (globoid cells), which tended to aggregate perivascularly. Gemistocytic astrocytes were abundant, and their nuclei were frequently abnormal. The activity of the deficient enzyme, galactosylceramide β-galactosidase, was undetectable in this neurologic disorder compared to age- and breed-matched control brains, and levels of the neurotoxic substrate, psychosine, were markedly elevated.

Globoid cell leukodystrophy (GLD) is a progressive and fatal lysosomal storage disease, which is classified as a sphingolipidosis. It is also termed Krabbe disease in recognition of the Danish neurologist who described the first human case in 1916.¹³ GLD is categorized as a leukodystrophy, a disorder that tends to develop early in life and involves selective and often bilaterally symmetrical damage to, and loss of, central nervous system white matter, much of which is the result of a heritable myelin defect.¹ GLD is caused by a deficiency of the enzyme galactosylceramide β-galactosidase, which results in impaired degradation and subsequent accumulation in the central nervous system of its major myelin lipid substrates, namely galactocerebroside and galactosylsphingosine (the latter also commonly designated as psychosine).^13,14

GLD in humans is usually an autosomal recessive disorder caused by mutations (>130 now identified) in the GALC gene, which has been mapped to human chromosome 14 at site 14q13.¹³ The infantile form of GLD is typically fatal before the age of 2 y, and manifests clinically as limb stiffness and seizures, progressing to severe motor and mental retardation.¹³ GLD has also been found in a number of dog breeds (in some of which an autosomal recessive mode of inheritance has been established), the domestic cat, the twitcher mouse (a naturally occurring mutant of GLD), and primates.¹⁷ However, our case is only the second to be described in sheep, to our knowledge, the first occurring in 2 Polled Dorset rams in Victoria, Australia in 1976.¹⁰

On a property in the Barossa Valley of South Australia, a 24-mo-old Merino wether was presented with a history of incoordination, muscle tremors and fasciculation of the head, neck, fore- and hindlimbs, and bruxism. This progressed to paralysis and sternal recumbency, with the hindlimbs extended to one side. The animal was eventually unable to stand and support its own weight. Over the past 5 y, 12 sheep on this farm had shown similar neurologic signs but, given that the occurrence was sporadic and confined to only a few animals, the cases had not been investigated further. We were unable to obtain any further clinical history on this flock and, accordingly, it is not known whether this GLD case had a heritable etiology.

An autopsy of our case was conducted by the referring veterinarian, and no gross findings were reported. Brain and cervical spinal cord were submitted fixed in 10% neutral-buffered formalin. Coronal sections of cerebral hemispheres, cerebellum, brainstem, and cervical spinal cord were cut at 5-mm intervals and embedded in paraffin; 6-µm sections were cut and stained with hematoxylin and eosin (H&E). Sections were also stained by periodic acid–Schiff (PAS), and Weil stain for myelin.

For immunohistochemical detection of astrocytic reaction (glial fibrillary acidic protein [GFAP]), microglia and macrophages (ionized calcium-binding adaptor molecule–1 [Iba-1]), and axonal injury (amyloid precursor protein [APP]), the following antibodies were used: rabbit polyclonal to GFAP (cat. Z0334, Dako, Carpinteria, CA), goat polyclonal to Iba-1 (cat. ab5076, Abcam, Cambridge, UK), and mouse monoclonal to APP (a gift from Dr. Colin Masters, University of Melbourne, Victoria, Australia), in a standard streptavidin-biotinylated immunoperoxidase technique. In brief, sections were dewaxed using xylene and rehydrated. Endogenous peroxidase activity was quenched in a 2% hydrogen peroxide–methanol solution for 30 min. After washing in phosphate-buffered saline (PBS) at pH 7.4, antigen retrieval was performed using citrate buffer (pH 6). Nonspecific binding was blocked using normal horse serum.

Antibodies to GFAP, Iba-1, and APP were applied at 1 in 40,000, 1 in 15,000, and 1 in 1,000, respectively. All antibodies were incubated overnight. The following day, all sections were given 2 washes in PBS, then biotinylated anti-goat secondary (cat. BA-9500, Vector Laboratories, Burlingame, CA) was applied to the Iba-1 sections, biotinylated anti-rabbit secondary (cat. BA-1000, Vector Laboratories) to the GFAP sections, and biotinylated anti-mouse secondary (cat. BA-2001, Vector Laboratories) to the APP sections. All of the sections were then incubated for 30 min at room temperature. Following the secondary incubation, 2 PBS washes were performed, and all slides were then incubated for 1 h at room temperature with a streptavidin-conjugate peroxidase tertiary reagent (cat. 21127, Pierce, Pasadena, CA). Sections were visualized using diaminobenzidine tetrahydrochloride (DAB), washed, counterstained with hematoxylin, dehydrated, cleared, and mounted on glass coverslips. A negative control omitting the primary reagent, as well as a positive control showing the normal pattern of expression of the antigen in question, was run with each batch of slides.

Small blocks of brain tissue were collected from the medulla at the level of the obex in our case, and from the same region in 3 age- and breed-matched control sheep. The tissues were resuspended in 0.02 M Tris (pH 7) containing 0.5 M sodium chloride and 0.1% nonidet P-40, and Dounce homogenized on ice. Total protein was determined by a published method,⁵ and single-phase lipid extraction was performed on 0.1 mg in 0.01 mL as reported previously.¹² Galactosylsphingosine was measured by liquid chromatography–electrospray ionization–tandem mass spectrometry in the same manner as described for glucosylsphingosine.³ Galactosylceramide β-galactosidase activity was determined in the same brain homogenates by measuring hydrolysis of the tritiated natural substrate (ceramide-[³H]-β-galactoside) producing the [³H]-galactose product as described previously.¹¹ The fresh liver copper level was 2.18 mmol/kg wet weight (normal range: 0.23–3.67 mmol/kg).

In our sheep, histologic brain lesions were most severe, and bilaterally distributed, in the cerebellar peduncles (Fig. 1A), but also were found in the cerebellar folia white matter, medulla (Fig. 1B), and pons. In the spinal cord, lesions were present in dorsomedial, ventromedial, and lateral funiculi.

Figures 1–4.

Globoid cell leukodystrophy in a Merino sheep.

Figure 1. A. Bilaterally distributed lesions in the cerebellar peduncles characterized by parenchymal pallor (H&E, left panel). B. Myelin loss, as demonstrated by Weil myelin stain (right panel). The sections of panel B show higher power views of a medullary lesion showing marked rarefaction of the neuropil. Figure 2. Perivascular aggregation of large globoid macrophages and a few multinucleate giant cells. H&E. Figure 3. A. Some perivascular macrophages are immunopositive to the macrophage marker Iba-1. B. Some macrophages contain PAS-positive storage material. Figure 4. Gemistocytic astrocytes, many with abnormal nuclei in a cerebellar peduncle. H&E. Inset: large GFAP-immunopositive astrocytes are shown.

Microscopically, affected areas in H&E-stained sections were very pale-staining and sometimes well-demarcated, especially in the cerebellar peduncles. Much of this pallor was the result of myelin loss, as demonstrated by Weil myelin staining (Fig. 1). The neuropil was rarefied. The most prominent histologic feature, apart from marked myelin loss, was the presence of numerous, large, ovoid macrophages with abundant cytoplasm (so-called “globoid cells”), either dispersed individually or in small clumps throughout the affected neuropil or, as distinctive perivascular cuffs (Fig. 2). However, the endothelium of such vessels appeared generally non-reactive. Many multinucleate giant cells were also found. The cytoplasm of these macrophages was ample and eosinophilic, often finely granular and, less commonly, contained ill-defined paler areas or, occasionally, vacuoles. A substantial subset of these macrophages and multinucleate giant cells showed strong cytoplasmic immunopositivity to the macrophage and microglial marker Iba-1 (Fig. 3A), and a small amount of PAS-positive material was demonstrable in these macrophages (Fig. 3B).

Gemistocytic astrocytes were a prominent histologic finding in the white matter, their nuclei often being large and appearing abnormal (Fig. 4). These nuclei were variable in size and shape and contained coarse, chromatin clumps and sometimes inclusions resembling cytoplasmic invaginations (Fig. 4). In GFAP-immunostained sections, there was diffuse astrogliosis in affected areas, with gemistocytic astrocytes with abundant, GFAP-immunoreactive, intermediate filament-containing cytoplasm (Fig. 4, inset). Glial cells with oligodendrocyte morphology were reduced in number as qualitatively assessed. A few APP-immunopositive axonal swellings (spheroids) were found, but their numbers were small.

GALC activity in our case was 0 pmol/min/mg; GALC in the 3 control brains was 0.5, 0.5, and 0.9 pmol/min/mg, respectively. The psychosine level in the GCL case was 5.6 nmol/mg; in the control brains, the level was 0.04 nmol/mg in all 3 sheep.

The previously reported ovine cases of GLD were in 2 Polled Dorset rams (4 and 18 mo old) on a property in western Victoria, Australia.¹⁰ Clinical signs and distribution of brain lesions were similar to those found in our case. The younger lamb had hindlimb incoordination, which eventually progressed to tetraplegia. The lamb was hyperesthetic, could not stand unassisted, and had a slight head tremor; withdrawal reflexes were depressed, placing reflexes were absent, and the patella tendon reflex was exaggerated. Our older animal had hindlimb ataxia, hypermetria, and stumbling, and the reflex pattern resembled that of the lamb. Given that demyelination was a major histopathologic feature, liver copper levels were analyzed and found to be substantially lower than unaffected control sheep; by contrast, the liver copper level in our case was within the normal range. The low liver copper levels found in the previously reported GLD cases could potentially have contributed to the myelin deficiency, particularly given that the 4-mo-old lamb was within the age range in which enzootic ataxia can occur. However, other sheep in the same region also had low liver copper levels and did not show signs of enzootic ataxia, and it was considered unlikely that enzootic ataxia and GLD would occur concurrently. GALC was deficient in both brains. The parents of the 2 sheep autopsied could not be identified.¹⁰

With respect to differential diagnoses, fibrinoid leukodystrophy (Alexander disease) has been categorized as a leukodystrophy in Merino sheep¹ but, although myelin loss is a feature of some forms of this disorder in human and domestic animals, it was minimal in 3 Merino sheep with this neurologic disease.⁶ Moreover, Alexander disease is characterized by the formation of signature intra-astrocytic Rosenthal fibers, which have not been described in GLD.^13,14 Myelin deficiency is also found in copper-deficient sheep (enzootic ataxia) up to ~6 mo of age.⁷

Pathologic changes in human GLD are confined to the central and peripheral nervous systems. In the most common infantile form, the brain is macroscopically atrophic, and hallmark microscopic changes are a marked paucity of myelin (with some attendant axonal degeneration), extensive fibrillary gliosis, infiltration of numerous, large, round macrophages (globoid cells), some of which are multinucleate, and markedly reduced numbers of oligodendrocytes. The characteristic globoid cells, which contain PAS-positive storage material, are most abundant in regions of active demyelination and often cluster around blood vessels; storage material may also be found in neural cells, particularly oligodendrocytes and Schwann cells. There is some evidence that activation of microglia (and astrocytes) precedes oligodendroglial and myelin loss, and that globoid cells may represent activated microglia, which then damage oligodendrocytes.^8,9 Although astrocytic gliosis has been described previously in human¹³ and ovine¹⁰ GLD cases, the large, atypical astrocytic nuclei found in our case were not specifically commented upon in the former cases.

In the normal brain, GALC substrates are processed by the lysosome, and recycled compounds are able to enter the remyelination pathway. In GLD, mutations result in diminished GALC enzyme activity and impaired degradation of lipids during myelin turnover. Remyelination is much less effective, and myelin turnover continues, leading eventually to marked myelin loss. In spite of the fact that galactosylceramide is the principal substrate of GALC, it does not accumulate in the brain to any significant extent. The very rapid, and early, loss of myelin-forming oligodendrocytes markedly depletes the synthesis of galactosylceramide and limits its accumulation to characteristic globoid cells. In the first year of life, maximal synthesis and turnover of myelin normally occurs, attended by peak GALC activity. Myelination then slows and, in the adult, myelin is relatively stable and turnover is minimal.¹³

In contrast to galactosylceramide, which is not considered to be particularly injurious, there is a large accumulation of the toxic metabolite, psychosine, which plays a key role in degeneration of oligodendroglia and myelin. In our case, the psychosine level was markedly elevated compared to control brains. Although the pathogenesis of GLD has not been fully elucidated, psychosine could trigger membrane destabilization, leading to cell lysis, and its accumulation has been linked to oxidative stress, mitochondrial dysfunction, apoptosis, inflammation, and neuronal and axonal damage.^4,16 In humans, accumulated psychosine levels show a 100-fold increase in the GLD nervous system^15,17 and correlate biochemically with diminished GALC enzyme activity. Although histopathology is an important diagnostic modality, GLD in humans is currently diagnosed by a low GALC enzymatic assay and genetic mutation analysis, in concert with characteristic neurologic signs.^4,8 Patients with a GALC enzymatic activity of <5 nmol/h/mg of protein are considered to be at risk for development of GLD.²