Fabry Disease: Immunocytochemical Characterization of Neuronal Involvement

Gabrielle A. deVeber, MD," Gerald A. Schwarting, PhD,i Edwin H. Kolodny, MD,* and Neil W. Kowall, MD*

~ ~

Fabry disease is an X-linked glycosphingolipid storage disease caused by deficiency of a-galactosidase. Storage of globotriaosylceramide, also known as ceramide trihexoside, is maximal in blood vessels but also occurs in neurons. We performed neuropathological histochemical studies on the brains and spinal cords of 2 patients with confirmed Fabry disease. Luxol fast blue-positive deposits were found in blood vessels throughout the central and peripheral nervous system and within selected neurons in spinal cord and ganglia, brainstem, amygdala, hypothalamus, and entorhinal cortex. Regions adjacent to involved neuronal groups, including nucleus basalis, striatum, globus pallidus, and thala- mus, were spared. Electron microscopy showed lamellar cytoplasmic neuronal inclusion bodies. Using a monoclonal antibody reactive with ceramide trihexoside, we found more extensive neuronal deposition than evident by Luxol-fast blue staining and new areas of neuronal storage in the spinal cord and cerebral cortex. Blood vessels throughout the nervous system were strongly immunoreactive. The highly selective pattern of neuronal involvement we found sug- gests that glycosphingolipid exposure, uptake, or catabolism varies greatly with respect to neuronal morphology and distribution. The degree of toxicity to neurons and the clinical significance of this neuronal storage remains to be defined.

deVeber GA, Schwarting GA, Kolodny EH, Kowall NW. Fabry disease immunocytochemical characterization of neuronal involvement. Ann Neurol 1992,3 1 409-4 15

Fabry disease, an X-linked deficiency of a-galactosi- dase C1, 21, is characterized by excess lysosomal stor- age of specific glycosphingolipids, predominantly cera- mide trihexoside (CTH) and digalactosylceramide, also known as ceramide dihexoside (CDH). Clinical fea- tures primarily result from angiopathy due to storage of glycosphingolipids in vessel walls 121. Neuronal ac- cumulation of lipid-like material in specific subsets of neurons has been delineated using traditional Luxol- fast blue (LFB) and other cytological staining methods r3-91. Studies of brain homogenates document in- creased levels of C T H and C D H throughout the brain 17-1 11. The presence of high levels of CTH in extra- neuronal elements within brain homogenates, how- ever, precludes the direct determination of neuronal CTH . To define further cht: nature and extent of the glyco-

lipid accumulation in Fabry disease, we studied the dis- tribution and identity of the neuronal glycolipid using a novel monoclonal antibody specific for CTH in 2 patients with enzymatically confirmed Fabry disease. We compared the distribution of CTH immunoreactiv- ity to the distribution of glycolipid as seen with a tradi- tional lipid stain, LFB.

Patient Reports Patient 1 The patient initially presented with febrile episodes and pain- ful extremities as a child. He subsequently developed renal failure and was noted to have multiple skin angiokeraromas. During his last year of life he had three episodes of focal cerebral ischemia. He died of progressive renal failure at the age of 53. A younger brother had multiple cerebral strokes and died of renal failure at age 32. One daughter has episodic pains in her hands and feet. Skin fibroblast cultures showed low a-galactosidase activity, confirming the diagnosis of Fabry disease. Postmortem examination showed diffuse thickening and

yellow discoloration of blood vessel walls and several 2.5 to 10 mm lacunar infarcts in the right head of the caudate, right thalamus, right basis pontis, and left centrum semiovale. On microscopic examination, LFB-positive material was found within blood vessels throughout the brain and spinal cord and in a peripheral nerve sample. Granular LFB-positive cy- toplasmic material was seen within neurons in several areas, including the paraventricular and supraoptic nuclei of the hypothaiamus, basal nucleus of the amygdala, inferior tempo- ral and insular cortex, subiculum, substantia nigra, dorsal nucleus of the vagus, dorsal reticular nucleus, intermediolat- era1 cell column of the thoracic spinal cord, Onuf's nucleus of sacral cord, dorsal root ganglia, sympathetic ganglia, and

From Harvard Medical School and the "Department of Neurology, Massachusetts General Hospital, Boston; and the +Department of Biochemistry, E. K. Shriver Center. Waltham, MA. Received Jun 13, 1991, and in revised form Aug 28. Accepted for publication Sept 14, 1991.

Address correspondence to Dr deVeber, McMaster University Med- ical Centre, Department of Pediatrics, 3 N 11 J, 1200 Main St West, Hamilton, Ontario. Canada L8N 325.

Copyright G 1992 by the American Neurological Association 409

Meissner’s plexus. Ependymal cells and subependymal as- trocytes at times contained LFB-positive material, and LFB- stained macrophages were occasionally seen in the area post- rema, choroid plexus, and leptomeninges. Many cells in the adenohypophysis and glial cells in the neurohypophysis con- tained LFB-positive material. There was ne~ironal depletion in the dorsal root ganglia and degeneration of the dorsal columns and dorsal spinocerebellar tracts in the upper tho- racic cord and of lateral corticospinal tracts throughout the cord.

Putieiil 2 Since childhood the patient was unable to sweat and had febrile episodes with episodic painful paresthesias of the hands. H e had a hisrory of progressive renal failure and skin angiokeratomas but no known cerebral strokes. Fabry disease was diagnosed by greatly diminished leukocyte a-galactosi- dase levels. Several relatives were known to have Fabry dis- ease or to be carriers documented by biochemical studies. H is neurological examination was normal one week prior to death due to cardiac arrest at the age of 31 years. Postmortem examination showed LFB-positive material in

all systemic blood vessels and within cells of the renal glomer- uli and cardiac muscle. In the large bowel, ganglion cells of Meissner’s anci Auerbach‘s plexus and smooth muscle cells contained LFB-positive material. The whole and freshly cut brain and spinal cord were grossly normal in appearance. On microscopic examination, LFB-positive material was seen in endothelial cells ancl the media in blood vessels throughout the central anci peripheral nervous system. Microscopic in- farcts were seen in the head of the right caudate nucleus and in the right internal capsule. In the cerebellum, there was marked depletion of Purkinje cells. Hippocampal neurons in CA 1 were pyknotic with adjacent fibrillary astrocytosis. There was a patchy depletion of neurons throughout the cerebral cortex, and a mild astrocytosis and microglial prolif- eration were present in rhe fronroparietal cortex and white matter. consistent with hypoxic-ischemic eiicephalopathy. LFB-positive material was also seen in cells of the leptomen- inges, ependynial cells, and in the majority of neurons in the following locations: the paraventricular and supraoptic nuclei of the hypothalamus. the basal nucleus of the amygdala, the dorsal motor nucleus of the vagus and midline raphe nuclei of medulla, intermediolateral cell column of spinal cord, and dorsal root ganglia. Scattered positive neurons were seen in layers i and 6 of inferior temporal cortex, subiculum, sub- stantia nigra, nucleus ambiguus, anci intermdiolateral cell column of the thoracic spinal cord. Isolated pcisitive neurons were s ~ e n in the dorsal horn of thoracic and lumbar cord. Rare astroglial proliferation was evident in dorsal and inter- mediolateral columns of thoracic cord, and there was thin- ning of the neuropil in the intermediolateral tolumns.

Materials and Methods 7‘i.csue Prepurutiorz The brains from both patients were removed within 12 hours of death and fixed in formalin for standard paraffin embed- ding and LFB hematoxylin and eosin staining. Multiple tissue blocks representative of the entire neuraxis were also se- lected for cold fixation in periodate-lysine paraformaldehyde

for immunocytochemical studies. Samples from 3 control subjects without neurological disease and a patient with Alz- heimer’s disease were processed for immunocytochemistry in parallel with the tissue from the patients with Fabry discase.

Glycolipid A nalysii Monoclonal antibodies were produced by immunizing mice with PC12 cells as previously described [ 121. A clone, desig- nated “CC5,” produced an antibody that specifically recog- nized C T H on thin-layer chromatography (TLC.. I immuno- staining. Gangliosides were not immunolabelecl by CCS. Glycolipids were extracted from Fabry brain and normal brain tissue [ 131. and TLC immunostaining was perf(ormed { 12, 131. Glycosphingolipid immunochromatoRraphy was performed essentially as tlescribecl by Yamamoto and col- leagues [ 141.

Irnmunocytocbeniicul Pro~-edure.c Free-floating 50-pm frozen sections werc preinc ubated in absolute methanol and O.S(Y hydrogen peroxide for 3 0 min- utes, followed by washes in phosphate-buffered saline (PBS). Following 1 hour in lOC7 normal goat serum, section:. werc incubated overnight free-floating in a 1 : 10 dilution o f the anti-CTH immunoglobulin M (IgM) (CCS antibody) culturc supernatant in PBS with 0.3% Triton X- 100 tolloweil by washes in PBS. Sections were then incubated in antirnouse antibody IgM peroxidase conjugate ( 1 : 200 in PHS; Boeti- ringer Mannheim, Indianapolis, IN). Peroxiciase w a s dis- closed with a diaminobenLidine hydrochloride chromagen { 151. Some sections were counterstained with cresyl violet. The antibody also labeled CTH in sections of paraffin- imbedded tissue. Sections incubated in nonimniune .cerum did not contain any specific immunoreactivity.

Electron Micrompic Metbodc Blocks of tissue from Patient 1 were fixed in 4‘;( paraformal- dehyde and 257 glutaraldchyde, osmicated. dehydrateii, and embedded in plastic. Ultrathin sections were stxned with uranyl acetate and lead citrate and examined with a Phillipr electron microscope.

Results 1 ~ i ~ z A n o ~ ~ ~ t o c b e ~ i i . c t ~ i with C T H A&bodI Monoclonal antibody t o CTH (CIS antibody ) itid not stain cortical samples from any of the control brains (Fig 1A). In samples from both patients with Fhbry disease, however, blood vessels throughout thc brain and spinal cord were strongly immunoreactive (Fig lB), especially in the subpial vasculature. A gradient of intensity of staining surrounded some small vessels (Fig 1C). Immunohistochemical neuronxl staining was essen-

tially identical in tissue samples from the 2 patients with Fabry disease and closely adhered to the distt-ibu- tion of neurons staining with LFB (Fig 2). ;ilthough antibody staining showed m o r e extensive iieuronal involvement than LFB alone. Colocalization of LFB positivity with CTH immunoreactivity was consistently

410 Annals of Neurology Vol 3 1 No ’I April 1992

F i g 1. Ceramide trihexoside ICTH) vascular immunoreactioitj in the central nerz'ous system. (A! In noma1 adult human fron- tal cortex. C T H immunoreactivity is not detected. iimmunoper- oxidat. X 40.i (Bi In F a b v di.reaJe cortex. there is striking tni~iz~tioreacti~,it~ within blood i,essels (arrows) and in the nerd- ropil. linimuiioperoxidaJe. X 40.1 iC) HiRher magnt'jiration J.hous CTH inzmunoreactii~it~ ir7 endotheliul cells outlining a tmull blood wtsel in longitudinal sei-riot? (arrowheads). Another smull m r e l in cross section (asterisk) exhibits a decreasing gra- dient of inztnunoreactiz'it~ extending auu.y from the blood 1,esJ-d into the adjucent neziropil. Ilnimunoperoxiduse, x 400.)

Fig 2. Compurirori of Luxol-fast blue Jtaining t o crramide tt-i- hexoside ICTH, immunoreactii~itj in dor.rul root ganglion 111

Fad?? dijtuse. (A! Dorsal root ganglion neurons u?th iutenielj Luxol-fust blue-positiiie cytoplam (arrowheads) are inter- sperred among normal neurons u,ith eo.iinophilii. ~~topiu~rti (arrows). (Luxol-fast blue-hetnatoxq lin-eatin: x GO brfr;rc. 27'% reductiori.i tB1 Cl'H imtriuvioreactir,itj, hralizrd within the cytoplusnz ojsu.oi1en dorm' root guriglion neurons (arrow- heads 1 is morphologicalb identical t o neuronul stuining u itCi Luxol-fast blue i ~ i A. (Cresyl riolet counter.rtain: x 60 befire 27% reduction.!

found in double-stained sections (data not shown). As with LFB staining, neuronal groups strongly immuno- reactive in both patients with Fabry disease included specific nuclei within hypothalamus and amygdala, su- biculum, entorhinal cortex, substantia nigra (nonpig- mented neurons), dorsal motor nucleus of the vagus, midline raphe neurons in medulla, intermediolateral cell column, and dorsal root ganglion. In addition, in samples from both patients with Fabry disease, strongly positive neuronal antibody staining was seen in layers 5 and 6 of frontal, parietal, temporal, and occipital cor- tex (Fig 3A); substantia nigra pigmented neurons (Fig 4A); and anterior horn (Fig 4B) and dorsal horn neu- rons (substantia gelatinosa) throughout the spinal cord. Throughout the central nervous system (CNS), the highly selective pattern of neuronal involvement seen with LFB staining was maintained with immunohisto-

deVeber et al: Histochemistry of Fabry Disease 411

chemistry. For example, there was sparing of neurons in more superficial layers of cerebral cortex (see Fig 3A) and neurons in the amygdala outside of the heavily immunostained basal nucleus (see Fig 3B). As with LFB staining, neuronal groups in other areas of the CNS, including thalamus, caudate, putamen, and cere- bellum, did not show antibody staining. Within neu- rons, antibody staining was in a granular pattern, as seen with LFB staining, and was confined to the neu- ronal perikaryon and neuritic processes (see Fig 4'4,B). No nuclear staining was evident.

G/jliolipid rind) \ I I

The Fabry disease and normal brain glycolipid fractions were analyzed by TLC immunostaining with the CC5 antibody, which specihcally reacts with CTH and other alpha-galactose terminal glycolipids The major differ- ence between Fabry disease neutral glycolipids and normal brain neutral glycolipids was the presenc e of

C T H in the Fabry disease tissue, which was not seen in the normal brain (Fig 5). As shown in Figure SA, the CC5 monoclonal antibody reacted strongly with the C T H standard but not with the other standards, which have beta-galactosyl or beta-n;-acetylgalactosa- mine terminal sugars. CC5 antibody did not react with usual concentrations of any glycolipids in normal brain but reacted strongly with CTH in Fabry disease hain. In addition, several other minor glycolipicis in the Fabry disease brain tissue reacted with the CCS anti- body. CTH detected b y TLC (see Fig i B ) anJ TLC immunochromatography in the Fabry disease brain was not present in control subjects, which is consistent with our immunocytochemical result.

Electron Microscopj Ultrastructural study (only performed on Patient 1 ) re- vealed lamellar cytoplasmic inclusion boclies or "zehra bodies" in neurons of the supraoptic nucleus of the hypothalamus and dorsal root ganglion.

Discussion Cornpunion of Lud-fait Blur Stuiriiiig a i d Im m u no1 y toc hemi rhy The distribution of abnormal LFB-positicc neurons i n

our 2 patients agrees with previous description5 [ i - y ] Immunominiiig with the C T H Antibod\ showed that

F i g 4. Intraneuronal distribution of ceramide trihexoside (CTHi immunoreactiz'ity. iA1 Pigmented neurons in the sub- stantia nigra pars cornpacta contain C T H immunoreactizity, uihich extends into dendrites and axonal (arrow) processes. Mel- anin granules in the cell body are indicated bji the arrow head. (Crejyl d e t rounterstairi: X 640 before 24q reduction.) (Bi Sacral anterior ho rn w eu ro 71 (arrow j mil tat ns C T H immu n o re- ai-tiz.ity within the r-elf body and proximal detidrites. iCvesjd t i - olet counteritLn; x 640 behre 24V reduction.)

neurons not previously thought to be involved in Fabry disease (such as layers 5 and 6 of the neocortex [see Fig 3A), substantia gelatinosa, and motor neurons in spinal cord) accumulate CTH. In the substantia nigra only nonpigmented neurons were previously reported to store lipid [ 3 ] ; however, by antibody staining, pig- mented neurons were found to be CTH-positive. Neu- ronal lipid storage in cerebral cortex in Fabry disease was previously observed only in deep layers of tempo- ral and parahippocampal gyms [7), although biochemi- cal tissue assays have shown trace amounts [ 7 ] or high levels [S, 7, 161 of CTH in frontal cortex. These addi- tional areas of neuronal involvement suggest an in- creased sensitivity of antibody staining over traditional staining methods. The demonstration of intraneuronal deposits with a

specific antibody to CTH allowed direct identification of the LFB granular storage material. Lipofuscin is re-

CDH

CTH

1 2 3 1 2 3 F i g 5 . Thiw-layer chromatography (TLC) immunostaining and TLC of glyc-olipidJ. in Faby disease and normal brain. iA) Neutral glycolipids of nomzal human brain (lane 1 i , Fabqi brain (lane 21, arid glycolipid standards ilane 3 ) u ' e ~ e anahzed by TLC immunostaining with CC5 antibodji. CC5 antibody does not identify lipids in normal brain ilane 1 1 . ushereas in- tense immunoreactitity i s seen in Fabry disease brain ilane 21. zc6ich corresponds t o the ceramide trihexoside iCTHi standard in lane .3. Additiorial minor gi)colipids are also identzfied in Fabry disease brain. (Bi TLC of the same 3 samples in A are developed uith an orcinol spra-y reagent t o zmisualize neutral gh- colipidr. A zarietji of glycolipids are identi'jed in normal ilane 1 1 and Fabry disease (lane 2) brain. Note the intense staining of C T H in the patielit with Faby disease ilane 21. CDH = ceramide dihexosidr; C T H = ceramide trihexoside; GLOB0 = globotetraoalceramide: and AGM 1 = gang~iotetraosylceramide.

portedly stored in excess in some neurodegenerative disorders, including Fabry disease [S], but it was not CTH-immunoreactive in our control brains. Although excess CTH measured with biochemical means has been found in the same areas that exhibit abnormal neuronal LFB staining in Fabry disease [7], the pres- ence of high levels of CTH in blood, cerebrospinal fluid (CSF), and blood vessel walls (all necessarily con- tained in tissue blocks used for analysis) have pre- vented direct determination of neuronal CTH. The selectivity of the CC5 antibody, the consistent colocali- zation of LFB and antibody staining, the close relation- ship of immunostaining to the known distribution of lipid storage in Fabry disease, and the results of immu- nochromatography [see Fig 51 confirm the specificity of the antibody.

Mecbaniim of Neuvonal Glycolipid Storage The origin of the abnormal neuronal glycolipid is un- known. Endogenous site-specific neuronal production

deVeber et al: Histochemistry of Fabry Disease 413

of CTH has been proposed C27. Neutral glycolipids including CTH are normal membrane constituents in kidney, aorta, spleen, and liver C2], but concentrations of CTH in normal brain, spinal cord, and spinal ganglia have not been determined. We could onlv detect traces of CTH using concentrated glycoiipid extracts in nor- mal adult frontal cortex. Others have not detected CTH in normal brain C2, 111, although trace (<l%) levels of CTH were found throughout the CNS in one normal adult control subject 197. CTH and C D H have been found in normal human

fetal whole brain homogenates (17) and in Neimann- Pick disease type C 1181, another sphingolipidosis in which regional neuronal glycolipid storage occurs. The similar degree of neuronal C T H storage in our 2 pa- tients, despite an age difference of more than 20 years, suggests that the storage of excess CTH within neurons may stabilize in adulthood. Neuropathological studies have not been performed in infants or young children with Fabry disease, and the concentration of CTH dur- ing normal brain development has not been defined. If site-specific differences in C T H metabolism ac-

counted for the distribution of involved neurons, other unifying features of such neurons might be expected. N o cytological, anatomical, or functional feature distin- guishes CTH-positive neurons from neurons that are spared in Fabry disease. Magnocellular neurons of the basal nucleus of the amygdala and paraventricular and supraoptic nuclei of the hypothalamus are selectively involved, whereas smaller neurons in those structures are spared. Elsewhere, small neurons such as substantia gelatinosa interneurons in dorsal horn and small dorsal root ganglia neurons (19, 20) are preferentially in- volved. The putative neurotransmitters, functional af- filiation, and morphology of the involved neurons are highly varied. Absorption of high levels of CTH from blood has

been suggested as the source of glycolipid stored in endothelial cells and neurons, because circulating con- centrations of CTH are three- to ten-fold higher in patients with Fabry disease than in normal subjects (97. Anatomical location in areas of reduced blood-brain barrier 12 t] could potentially promote neuronal ab- sorption from blood, but not ail involved neuronal groups are found in such areas. Absorption of C T H from the CSF into adjacent neu-

rons could occur. Many of the involved neuronal groups are located adjacent to CSF, including hypo- thalamus, hippocampus, and certain dorsal brainstem nuclei. The selective sparing of neighboring neuronal groups, however, such as caudate, thalamus, and hypo- glossal nucleus, similarly exposed to CSF demonstrates that this cannot be the only mechanism. It is possible that subsets of neurons selectively take

up and transfer CTH. Neuronal uptake and transsyn- aptic transfer from peripheral to central neurons occurs

with herpes and rabies viruses and plasma proteins 1223. Such a mechanism of CTH transport could ac- count for the distribution of some neuronal groups involved in Fabry disease, such as transport from pe- ripheral autonomic to central autonomic neurons “)I; however, this mechanism does not account for thlt dis- tribution of others, such as the hippocampal neurons and amygdala.

Clinicopathologzral Correlation Correlation of neurological manifestations with neu- ronal storage of CTH is hampered by the presence of angiopathic infarcts and the widespread deposition of glycolipid throughout the central and peripheral ner- vous system and within end-organs such as the swear glands in the skin. The lancinating limb pain typical of Fabry disease

may be due to dorsal root ganglia neuronopathy [ 19 I or primary small fiber peripheral neuropathy 1211. His-- topathological studies have been inconclusive and con-- tradictory [ I 9, 23-27]. The involvement of substantia gelatinosa neurons we describe may contribute to the pathogenesis of painful limb paresthesias, although thc: more extensive involvement of dorsal root neurons is likely of greater importance. Autonomic dysfunction C28) could arise from involvement of the autonomic nervous system at either a central or peripheral level. The clinical correlate of the cerebral cortical giyco-

lipid deposition is unclear. Seizures are not a promi- nent feature of this condition, and electroencephalo-. gram recordings are usually normal [29}. Psyc.hosis, progressive personality disorder, and dementia have been reported in Fabry disease [4, 30,311. Multiple cerebral infarcts may obscure the interpretation of these manifestations. Detailed cognitive testing w ‘1s nor carried out on our patients, but they did not have a history of psychosis, dementia, or seizures. Given the paucity of clinical symptomatology related

to the accumulation of glycolipids in the CNS, it is possible that stored glycolipid is not neurotoxic The storage of excess CTH in endothelial cells and in 6- broblasts of the tunica media clearly produces an occlu-- sive angiopathy leading to infarction. It is not known if CTH storage in specialized cells of visceral organs independently contributes to organ dysfunction. L ~ c J - fuscin is thought to accumulate in aging neurons with- out causing degeneration [32], but this notion has been challenged [3 31. In summary, immunocytochemical investigation with

an antibody specific to C T H more thoroughly details the selective neuronal involvement in Fabry disease than traditional staining methods. The clinical ex- pression and pathophysiological mechanism of this highly selective neuronal involvement is not clear. Further studies of the developmental expression of specific glycolipids in the nervous system of both

414 Annais of Neurology Vol 31 No 4 April 1992

normal subjects and patients with Fabry disease are needed to clarify the mechanism and functional con- sequences of glycolipid accumulation within neurons in this condition.

This work was supported in part by National Institutes of Health Grants HD0055 15 (G.W.S.) and AG05134, NS10828, and NSZ5588 (N.W.K.). We would like to thank Karen Harrington, Robert Savio, Parker Ward, and Lawrence Cherkas for their excellent technical assistance. Also, we are grateful for details of the clinical history for Patient 2 supplied by Dr Gregory L. Braden at Baystate Medical Centre in Springfield, MA. We are especially grateful to Dr E. P. Richardson and D r J. P. Vonsattel for their expert help in reviewing the pathological material and their thoughtful comments.

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