Abstract Muscle biopsy tissue from a patient affected bythe juvenile form of neuronal ceroid lipofuscinosis (NCL)was studied immunohistochemically using antibodies toβ-amyloid peptide and amyloid precursor protein. Posi-tive reaction in muscle was specifically localized to au-tophagic vacuoles and blood vessel walls. Increased acidphosphatase reaction suggested enhanced lysosomal ac-tivity. We hypothesize that β-amyloid is deposited in NCLmuscle by a lysosomal mechanism similar to that pro-posed in other disorders involving β-amyloid.

Key words β-A4 amyloid · Amyloid precursor protein ·Juvenile neuronal ceroid lipofuscinosis

Introduction

Neuronal ceroid lipofuscinoses (NCL) are autosomal re-cessive neurodegenerative disorders of infancy or child-hood with a global incidence of 1 in 12,500 live births.Common to all NCL forms is the presence of intralysoso-mal autofluorescent lipopigments in several tissues, in-cluding neurons, leukocytes, and endothelial cells. Thesedeposits can have different ultrastructural features, vary-ing from granular structures to fingerprint or curvilinearbodies [14]. The three main clinical forms of NCL are

usually classified as infantile (Santavuori-Haltia disease),late infantile (Jansky-Bielschowsky disease), and juvenile(Spielmeyer-Vogt-Sjögren disease). The defective genesresponsible for the infantile and juvenile NCL have beenlocalized to chromosomes 1 [23] and 16 [18], respectively.More recently, the structural organization and sequence ofthe defective gene in classical late infantile NCL (CLN2)[22] and variant late-infantile NCL (CLN5) [21] have alsobeen identified. Here we report on a patient affected by ajuvenile form of NCL in whom the diagnosis was basedon the clinical picture, and on skin and muscle biopsyfindings. Since the composition of the lipopigments inbrains in juvenile NCL patients is complex and includessuch unusual components as β-amyloid [17] and amyloidprecursor protein (APP) [25], we also investigated thebiochemical composition of the autophagic vacuoles foundin the muscle of our patient by immunohistochemistrywith antibodies to β-amyloid filaments, APP, and ubiqui-tin. We also used antibodies against immunoglobulinlambda and kappa chains, against the neoantigens of thecomplement C5b-9 membrane attack complex (MAC) andagainst major histocompatibility complex class I mole-cules (MHC-I).

Case report

The patient was a 21-year-old male. The parents were healthy andnonconsanguineous. He was born after a normal pregnancy withan uneventful delivery. The neonatal period was unremarkable.Psychomotor development was normal up to age 6 years. While at-tending primary school, he started to complain of visual failure.The consulted ophthalmologist diagnosed a macular degeneration.A neurological examination at this age was normal. Visual lossgradually worsened, with another funduscopic examination at age14 showing papillary atrophy. At age 17, the patient was admittedto a neurological clinic because of progressive gait and speech ab-normalities associated with generalized seizures and mental retar-dation. Speech was monotonous. EEG showed generalized epilep-tiform discharges with abnormal background activity. Computedtomography (CT) showed dilatation of the fourth ventricle. Pheno-barbital was administered leading to complete seizure control. Thepatient was brought to our attention at the age of 20 due to pro-gressive deterioration of his clinical condition. Generalized seizureshad become frequent, and administration of various anti-epileptic

M. Villanova · C. Ceuterick · M. T. Dotti ·F. M. Santorelli · C. Casali · A. Malandrini ·N. De Stefano · U. Lübke · J. J. Martin · G. C. Guazzi ·A. Federico

Detection of â-A4 amyloid and its precursor protein in the muscle of a patient with juvenile neuronal ceroid lipofuscinosis (Spielmeyer-Vogt-Sjögren)

Acta Neuropathol (1999) 98 :78–84 © Springer-Verlag 1999

Received: 19 August 1998 / Revised: 16 November 1998 / Accepted: 18 November 1998

REGULAR PAPER

M. Villanova · A. Malandrini · G. C. GuazziLaboratory of Neuropathology, Institute of Neurological Sciences, University of Siena, Siena, Italy

C. Ceuterick · U. Lübke. J. J. MartinLaboratory of Neuropathology, Born-Bunge Foundation, University of Antwerp, Wilrijk, Belgium

M. T. Dotti · N. De Stefano · A. Federico (Y)Neurometabolic Unit, Institute of Neurological Sciences, University of Siena, I-5100 Siena, Italye-mail: Federico@unisi.it, Fax: +39-577-40327

F. M. Santorelli · C. CasaliLaboratory of Genetics, Institute of Neurological Sciences, University “La Sapienza”, Rome, Italy

drugs could not completely control the seizures. Neurological ex-amination showed severe mental retardation, diffuse muscle hypo-tonia, ataxic gait, visual loss and areflexia. The convulsive disor-der was associated with myoclonus of the arms. EEG showed sig-nificant deterioration compared to the previous one performed atage 17. CT scan and magnetic resonance imaging showed general-ized cerebral atrophy, and even more severe atrophy of the cere-bellum. An important dilation of the fourth ventricle concomitantwith enlarged cerebellar sulci was observed. Needle electromyog-raphy (EMG) showed neurogenic changes. Measurement of sen-sory nerve action potentials and conduction velocity was normal.Visual studies showed abnormalities of the retinal pigment epithe-lium associated with electrophysiological (ERG) changes. Meta-bolic disorders able to cause myoclonus epilepsy and mental retar-dation were excluded by biochemical studies. A muscle biopsywas taken from the left vastus lateralis, with the overlying skin.

Materials and methods

Tissue

Muscle and skin biopsies were taken from the left vastus lateralismuscle. Prior to immunohistochemical study, the muscle biopsysample was examined with standard histological staining tech-niques, including hematoxylin-eosin, trichrome Masson, periodicacid-Schiff (PAS), PAS following amylase treatment, Sudan, andenzyme histochemical techniques for succinate dehydrogenase(SDH), NADH-tetrazolium reductase (NADH-TR), menadione α-glycerophosphate dehydrogenase, cytochrome c oxidase (COX),phosphorylase, and ATPase [6]. Histochemical methods were alsoused to detect acid phosphatase [2].

Control tissue was obtained from three patients without de-tectable muscle disease, three patients with sporadic inclusionbody myositis (IBM), two patients with oculopharyngeal musculardystrophy (OPMD), and two patients with polymyositis with se-vere structural muscle changes.

Immunocytochemistry

The muscle biopsy samples were snap-frozen in liquid nitrogen-cooled isopentane, and 7-µm-thick cryostat sections were cut.Nonspecific immunoglobulin binding sites in the tissue wereblocked by incubation of the sections with 1:25 normal swineserum (Dakopatts, Glostrup, Denmark) in 0.05 M TRIS-bufferedsaline (TBS) pH 7.4, containing 1% bovine serum albumin fractionV (BSA). Sections were then incubated overnight with primary an-tibodies. The antibodies and dilutions used are listed in Table 1.Immunostaining was performed by the avidin-biotin-complex(ABC) or the peroxidase-antiperoxidase (PAP) [15] methods. Sec-tions were photographed in a Reichert Polyvar optical microscope.

Negative controls consisted of preincubation with TBS and 0.1%BSA, incubation with non-immune mouse IgG, or omission of theprimary antibody.

Autofluorescence

To evaluate autofluorescence, a frozen muscle section from theNCL patient and from an aged control were examined under aZeiss Axioplan fluorescence microscope using an ultraviolet filterwith excitation bandpasses of 340–380 nm.

Electron microscopy

For electron microscopic examination, muscle and skin tissue fromour patient was fixed in 4% glutaraldehyde and postfixed in 2%osmium tetroxide, dehydrated in graded alcohols and embedded inAraldite (Fluka, Buchs, Switzerland). Ultrathin sections werestained with 2% uranyl acetate and lead citrate and observed in aPhilips CM 10 electron microscope at 60 kV. A pellet of lympho-cytes, isolated from our patient according to established tech-niques, was also studied by electron microscopy.

Molecular genetics

Using oligonucleotide primers BD1F and BD2R, we tested leuko-cyte total DNA for the presence of the 1.02-kb deletion in theCLN3 gene, as previously reported [26]. This genomic deletionhas been determined to be a common mutation, encountered inabout 70% of Batten disease chromosomes.

Results

Light microscopy

The muscle biopsy showed an overall myopathic aspect,with increased variation in fiber size and both atrophicand, more rarely, hypertrophic muscle fibers. Rare inter-nalized nuclei were also observed. Regenerative musclefibers or necrotic fibers undergoing phagocytosis wereonly rarely seen. A number of fibers appeared to be vac-uolated (Fig. 1A). Some of these vacuoles were rimmed(Fig. 1B). The NADH-TR, menadione α-glycerophos-phate dehydrogenase, SDH, COX, and ATPase prepara-tions showed focal loss of activity (Fig. 1C, D). However,a two fiber pattern with slight type 1 predominance wasretained. The PAS staining was positive in many vacuoles,though some vacuoles remained negative. Several vac-uoles showed an increased Sudan reaction (Fig. 1E).There was strong acid phosphatase activity throughout themuscle tissue (Fig. 1F).

Immunohistochemistry

Positive immunostaining was found around and insidemany vacuoles with antibodies against β-amyloid andAPP (Fig. 2A, C). Endomysial capillaries and large per-imysial blood vessels were also positive for these antibod-ies (Fig. 2B). The antibody against ubiquitin also stainedsome vacuoles. Anti-desmin antibody diffusely stainednormal muscle fibers, representing the normal reactivity

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Table 1 Antibodies used in this study (pAb polyclonal antibody,mAb monoclonal antibody, APP amyloid precursor protein, MACmembrane attack complex, Ig immunoglobulin)

Antibody Dilution Clonality Source

Ubiquitin 1/200 pAb Dakopattsβ-A4 amyloid 1/500 pAb Prof. BeyreutherAPP 22C11 1/30 mAb Boehringer MannheimDesmin 1/100 mAb DakopattsHLA-ABC 1/100 mAb DakopattsMAC 1/100 mAb DakopattsIgG 1/200 mAb DakopattsIg λ chain 1/100 mAb DakopattsIg κ chain 1/200 mAb Dakopatts

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Fig.1 Cryostat sections of NCL muscle biopsy stained withhematoxylin-eosin A, trichrome Masson B, NADH-TR B, ATPase

10,4 D; Sudan E, acid phosphatase F (NCL neuronal ceroid lipo-fuscinosis). A–F × 190

of the intermyofibrillar network, but a stronger reactionwas found in vacuolated muscle fibers (Fig. 2D). No de-posits of MAC, IgG or immunoglobulin light chains wereobserved. Normal immunostaining was found with the an-tibody against HLA-ABC.

Control tissue

In muscle biopsy specimens from patients affected byOPMD and IBM, the wall and sometimes the center of therimmed vacuoles were immunolabeled with antibodies toAPP and β-amyloid. With the antibody against HLA-ABC, all muscle fibers in muscle biopsies from patientswith polymyositis showed a significant sarcolemmal im-munostaining. Most of the necrotic fibers were immuno-labeled by antibodies against immunoglobulin lightchains. Few of them were MAC positive. This pattern ofimmunostaining was completely absent in muscle biopsyspecimens from patients without detectable muscle dis-

ease. No immunostaining was observed with omission ofthe primary antibody. After staining with non-immunemouse IgG, a weak stain was observed in the endomy-sium, but no other structures were labeled.

Autofluorescence

A considerable increase in autofluorescent material wasobserved in the sarcoplasm of the muscle fibers in our pa-tient compared to aged control tissue.

Electron microscopy

Muscle

Numerous inclusions (1–1.5 µm) with curvilinear and rec-tilinear profiles were observed in the subsarcolemmal areaand the intermyofibrillar network. Numerous vacuoles con-taining lamellar curvilinear and rectilinear aggregates werealso observed (Fig. 3). These vacuoles also showed occa-sional myelin-like figures. Non-filamentous material wasalso seen. Numerous osmiophilic granular and amorphousinclusions were also found in the endomysial fibroblasts.

Skin

Numerous inclusions with curvilinear, rectilinear and fin-gerprint profiles were observed in smooth muscle cells(Fig. 4), eccrine sweat glands and Schwann cells.

Pellet of lymphocytes

Membrane-bound vacuoles containing inclusions with fin-gerprint profiles, were encountered in some lymphocytes.

Molecular genetics

Employing a PCR-derived method, we did not detect thecommon 1.02-kb CLN3 deletion in our patient.

Discussion

In the present study, we immunocytochemically examineda muscle biopsy specimen of a patient affected by a juve-nile form of NCL. The diagnosis of NCL in our patientwas made by clinical and morphological studies. In mole-cular genetic studies we did not detect the common 1.02-kb CLN3 deletion in our patient [26]. These results, how-ever, cannot rule out that other, less common CLN3 muta-tions could have produced this patient’s neurodegenera-tive illness. The diagnosis of NCL is usually made by skinbiopsy and, probably for this reason, muscle tissue hasrarely been studied [5, 10]. Many hypotheses of its patho-genesis have been advanced, but relatively few are still

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Fig.2 Cryostat sections of muscle stained with antibodies to β-A4A showing positive staining in two vacuoles (arrows) and B in per-imysial blood vessel walls; C APP (22C11) showing granular im-munoreactivity in a vacuole; D desmin, showing stronger reactionin vacuolated muscle fibers (APP amyloid precursor protein). A × 312; B, C × 324; D × 220

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currently entertained [9]. The composition of the lipopig-ments characteristic of NCL is highly complex, butamongst others includes amyloid proteins. Amyloid is ageneric term used to designate proteins of diverse originsthat are often biochemically unrelated, but invariablyform fibrillar deposits by aggregation of relatively smallpeptides in a characteristic cross-β conformation [7, 8].

These amyloid peptides are usually formed by the enzy-matic processing of a precursor protein (amyloidogenicprotein). The nature of the precursor protein may vary,from immunoglobulin light chains [8] through variousserum proteins [3, 12] to APP [16]. We have found no ev-idence that immunoglobulin light chains were involved inthis case. On the other hand, β-amyloid has been previ-ously demonstrated in storage pigments in neurons ofNCL patients [17]. In that study, β-amyloid was ultra-structurally immunolocalized to curvilinear and finger-print profiles in affected neurons. However, these authors

Fig.3 Electron microscopy of NCL muscle biopsy specimen.Mixed curvilinear and rectilinear lamellar profiles within an in-termyofibrillar vacuole. × 61500

did not study any muscle tissue. In our present study ofNCL muscle we were able to show β-amyloid and APPimmunoreactivity in autophagic vacuoles and blood ves-sel walls, which corresponded ultrastructurally to lipopig-ment inclusions with curvilinear and rectilinear profiles.

β-Amyloid has previously been described in other dis-eases: Alzheimer’s disease (AD), where it forms the coreof senile plaques; and several muscle disorders, includingOPMD [24] and IBM [1, 19, 24], where it is specificallyassociated with rimmed vacuoles. These data are con-firmed in this study. The rimmed vacuoles have been alsoshown to be positive for the lysosomal proteases cathep-sin B and D [24], which suggests a lysosomal involve-ment in the processing of β-amyloid in these disorders.Lysosomal processing of β-amyloid has also been hypoth-

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Fig.4 A Electron microscopy of NCL skin biopsy specimen. Ar-teriolar smooth muscle cells displaying two membrane-boundpleomorphic inclusions (arrows) with fingerprints. B Higher mag-nification of the typical fingerprint profiles. A × 10 875; B × 61500

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esized to play a role in AD neurodegeneration [4, 11, 13].Furthermore, the strong acid phosphatase reactivity ob-served in the muscle of our NCL patient suggests a highlysosomal activity. Taken together, these results suggestthat β-amyloid and APP deposition in NCL muscle maybe determined by lysosomal activity, as seems to be thecase for other muscle and neurological diseases involvingβ-amyloid and APP. Defective lysosomal processing of β-amyloid and other proteins may be an important factor inlipofuscinogenesis in NCL, as already postulated for AD[4, 11, 13] and aging [20]. Thus, altered regulation of APPprocessing in the lysosomal system could also representan aspect of the pathogenesis of NCL. Further study willbe needed to elucidate this hypothesis.

Acknowledgements We thank Roberta Salvestroni for excellenttechnical work. We also thank Marie Louise Basso for editing themanuscript.

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