Acta Neuropathol (1999) 97 : 469–480 © Springer-Verlag 1999 R E G U L A R PA P E R D. Rodriguez · A. Gelot · B. della Gaspera · O. Robain · G. Ponsot · L. L. Sarliève · S. Ghandour · A. Pompidou · A. Dautigny · P. Aubourg · D. Pham-Dinh Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases Received: 23 February 1998 / Revised: 12 August 1998, 20 October 1998 / Accepted: 21 October 1998 Abstract We report neuropathological, biochemical and molecular studies on two patients with childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome, a leukodystrophy recently defined according to clinical and radiological criteria. Both had severe cavitating orthochromatic leukodystrophy without atrophy, predominating in hemispheric white matter, whereas U-fibers, internal capsule, corpus callosum, anterior commissure and cerebellar white matter were relatively spared. The severity of white matter lesions contrasted with the rarity of myelin breakdown products and astroglial and microglial reactions. In the white matter, there was an increase in a homogeneous cell population with the morphological features of oligodendrocytes, in many instances presenting an abundant cytoplasm like myelination glia. These cells were negative for glial fibrillary acidic protein and antibodies PGM1 and MIB1. Some were positive for myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, but the D. Rodriguez (쾷) · G. Ponsot · P. Aubourg Service de Neuropédiatrie, Hôpital Saint Vincent de Paul, AP-HP, 74–82 avenue Denfert-Rochereau, F-75674 Paris Cedex 14, France e-mail: drodrig@infobiogen.fr, Fax: +33-1-40-48-83-56 D. Rodriguez · P. Aubourg Pathologie métabolique et hormonale du développement, INSERM U342, Hôpital Saint Vincent de Paul, Paris, France A. Gelot · A. Pompidou Service d’Histo-Embryologie, Cytogénétique, Anatomopathologie, Hôpital Saint Vincent de Paul, AP-HP, Paris, France B. della Gaspera · A. Dautigny · D. Pham-Dinh Laboratoire de Neurogénétique Moléculaire, CNRS URA 1488, Université de Paris VI, Paris, France O. Robain INSERM U29, Hôpital Port-Royal, Paris, France L. L. Sarliève · S. Ghandour Laboratoire de Neurobiologie Moléculaire des Interactions cellulaires, CNRS UPR 416, Université Louis Pasteur, Strasbourg, France majority were positive for human 2′-3′ cyclic nucleotide 3′ phosphodiesterase and all were positive for carbonic anhydrase II, confirming that they are oligodendrocytes. Myelin protein and lipid content were reduced. The PLP gene, analyzed in one case, was not mutated or duplicated. The increased number of oligodendrocytes without mitotic activity suggests an intrinsic oligodendroglial defect or an abnormal interaction with axons or other glial cells. This neuropathological study supports the notion that CACH syndrome constitutes a specific entity. Key words Leukodystrophy · Oligodendrocyte · Myelin · Orthochromatic · CACH syndrome Introduction A large proportion of childhood leukodystrophies, most of which are genetically inherited, have no known etiology [15]. Phenotypic classification of these myelin disorders is essential for linkage analysis in affected families. A new leukoencephalopathy termed childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome or leukoencephalopathy with vanishing white matter was recently identified on clinical and neuroradiological grounds [13, 33, 34, 38]. This disorder only affects myelin of the central nervous system (CNS) and has an autosomal recessive mode of inheritance, but the genetic defect is not known. Previous neuropathological reports of two brain biopsies and one autopsy showed extensive cavitating leukoencephalopathy but with no significant histological findings [34, 38]. Here, we report neuropathological data of two new patients with CACH syndrome and a biochemical and molecular study of one of them. The cavitating white matter lesions which characterize this orthochromatic leukodystrophy are associated, in the less affected white matter areas, with an unusually increased oligodendrocyte density. 470 Materials and methods Clinical data Patient 1 This male patient had a normal family history with healthy, nonconsanguineous parents and an unaffected 14-year-old sister. He learned to walk at 15 months but speech was mildly delayed. The first disabilities in walking were noted after a minor head trauma when he was 7.5 years old. He was mildly macrocephalic (head circumference at +2 SD, like his father). Neurological examination showed cerebellar ataxia and diplegia. Computed tomography scan and magnetic resonance imaging (MRI) showed diffuse abnormalities of the cerebral white matter. Hemispheric white matter signal intensity was low on T1-weighted images, and partially low and similar to the CSF signal on proton density images. On T2-weighted images, signal intensity of hemispheric and cerebellar white matter was homogeneously increased with two particularly intense spots in the pontine tegmentum. U-fibers were spared. Visual- and somatosensory-evoked potentials showed a decrease in central conduction. Brain stem auditory potentials, motor and sensory peripheral nerve conduction and electromyogram were normal. Laboratory tests were negative for all peroxysomal, lysosomal and mitochondrial leukodystrophies. N-Acetylaspartic acid, oligosaccharides, amino acids, organic acids and sialic acids were normal in urine. The karyotype was also normal. The disease was characterized by progressive deterioration with optic atrophy, seizures, vomiting and bulbar symptoms. At age 10, the patient became comatose after a minor head trauma and died 2 months later. common to proteolipid protein (PLP) and DM20 [28] and an antirat carbonic anhydrase (CA) II antibody diluted 1:200 [11] were used. Astrocytes were stained with mouse anti-human glial fibrillary acid protein (GFAP; Dakopatts, Denmark) diluted 1:25. Microglia-macrophages were stained with a monoclonal mouse antihuman CD68 IgG3, PGM1 (Dakopatts) diluted 1:100 [16]. Mitotic activity was detected with a mouse monoclonal antibody against nuclear antigen 2KI67, MIB1 (Immunotech, France) diluted 1:20. A 13-week-old fetus was used as a positive control. The Vectastain avidin-biotin-peroxidase system (Vector laboratories) was used for product detection. Some slides were double immunostained to identify the mitotically active (anti-MIB1/anti-GFAP, anti-MIB1/ anti-PGM1, anti-MIB1/anti-CNPase) and hypercellularity (antiGFAP/anti-PGM1) cell types, revealed with peroxidase and alkaline phosphatase. Cell counts were performed on matched brain specimens from patients and a control who died at the age of 10 years from LeshNyhan disease. Using an ocular net micrometer with 100 squares, total cell number, with the exception of vascular cells, was counted by the same person, in comparable areas of white matter, on paraffin-embedded slides stained with CV [36, 39]. Labeled cells were also counted after GFAP, PGM1, CNP, CAII, and MIB1 immunostaining. Five counts were made for each of the values reported in this study. Results are expressed as the mean for each sample. Biochemical and molecular analyses Neuropathological analysis Postmortem cerebral and cerebellar white matter from patient 1 and from the Lesh-Nyhan patient were used to prepare total homogenates or to isolate myelin. Myelin was purified according to Norton and Poduslo [26]. Total lipids and myelin lipids were extracted [8] from the cerebellum and measured by weighing the dried lipid extract. Polar lipids, galactolipids and phospholipids were separated by high performance thin-layer chromatography (HPTLC) and quantified as described [14]. UDP-galactose:ceramide-galactosyltransferase (EC 2.4.1.45, CGalT) was assessed in brain homogenates [24]. One unit corresponds to 1 nmol of galactose transferred per hour. Total cerebellar homogenates were used for SDS-PAGE and immunoblotting [17]. PLP immunoblotting was performed as described [28]. Total RNA extracted from cerebral white matter was reverse-transcribed as previously described [29] according to standard procedures [32]. The coding sequence of the PLP cDNA was subsequently amplified using specific primers (the sequence of PLP-specific primers can be obtained upon request), and sequenced using the T7 sequencing kit (Pharmacia Biotech). Southern blots were hybridized with a 32P-labeled human PLP cDNA probe. For quantitative analysis, PLP signals were compared to signals obtained by hybridization with the XV2 C probe, an internal marker of the X chromosome. The brains were removed immediately after death and quickly fixed or frozen in cooled isopentane. Several fragments were fixed in 4% paraformaldehyde-1% glutaraldehyde in phosphate buffer, post-fixed in 4% osmium tetroxide, embedded in epoxy and cut on an ultramicrotome in semithin and thin sections for ultrastructural analysis (Philips CM10). The remaining fragments were fixed in formalin, embedded in paraffin or frozen after immersion in a 10% sucrose solution, and cut into 8-µm sections. Paraffin sections were stained with hematoxylin and eosin (H&E), cresyl-violet (CV), Luxol-fast-blue (LFB), periodic acid-Schiff (PAS) or silver impregnated by the Bodian technique. Frozen fixed sections were used for lipid revelation (Oil-Red-O, ORO) and immunostaining. Specific CNS cell types were identified using the following primary antibodies. Oligodendrocytes and myelin were stained with a monoclonal mouse anti-human myelin basic protein (MBP) IgG2 (Chemicon, Temecula, Calif.) diluted 1:50, a mouse monoclonal antibody against human 2′-3′ cyclic nucleotide 3′ phosphodiesterase (CNP; Chemicon) diluted 1:25, and a rabbit polyclonal anti-bovine myelin oligodendrocyte glycoprotein (MOG) serum [3]. On frozen tissues, an antibody against a C-terminal fragment Fig. 1A–F Telencephalic white matter. A Flair magnetic reso- 왘 nance image of patient 2 at age 5 years 9 months. Hemispheric white matter shows low signal intensity, similar to CSF, and is crossed by a meshwork of tissue, whereas signal intensity in the internal and external capsules is high. B Coronal section of the fixed brain of patient 2: white matter is diffusely cavitated, and appears flattened. There is no ventricular enlargement or thinning of the corpus callosum. C Closer observation reveals the radial cobweb pattern of remaining fiber bundles (case 2). D Macroscopic frontal section after MBP immunostaining showing widespread cavitation and preservation of subcortical myelin (case 2). E Frontal section revealing diffuse myelin depletion and relative preservation of perivascular myelin (case 1). F Sparse myelinated bundles in subcortical areas (top left). They become progressively disorganized when going deeper, except in perivascular areas (case 2) (MBP myelin basic protein). E, F Luxol-fast blue (LFB). E × 2, F × 10 Patient 2 This male patient had healthy unrelated parents and two healthy younger siblings. He walked at 14 months, but motor stagnation was noted at 16 months. Neurological examination showed spastic diplegia, cerebellar ataxia, but normal speech at the age of 2.5 years. Brain MRI showed abnormal hemispheric, cerebellar and brain stem white matter, as in patient 1 (Fig. 1A). Visual-, somatosensory- and brain stem auditory-evoked potential latencies showed delayed responses. Sensory and motor peripheral nerve conduction was normal. Biochemical tests for identified leukodystrophies were negative as in patient 1. The patient deteriorated slowly. He no longer walked at age 4, developed seizures and swallowing difficulties at age 5 and died at age 6 during a fever with recurrent apnea. Autopsy was unremarkable. 471 472 Table 1 Distribution and morphological features of white matter lesions (WM white matter, + present, ++abundant, +++ very abundant, – absent) Cerebral structures Cerebral central WM U-fibers, internal capsule Corpus callosum, anterior comissure, fornix, external capsule, cerebral peduncles Cerebellar WM Nucleus dentatus hilus Superior cerebellar peduncle Superior cerebellar peduncle at decussation level Tractus tegmentalis centralis Pyramidal tract Olivary nucleus (hilus) Inferior cerebellar peduncle Medial cerebellar peduncle, lemniscus medialis, cranial nerve Macroscopic study Myelin aspect Microscopic study Myelin loss Axon loss Hypercellularity Macrophages fully filled by sudanophilic myelin debris Cavitary (persistent perivascular myelin) Slightly discolored Slightly discolored +++ + – ++ ++ ++ – – ++ – – – Slightly discolored Grayish aspect Discolored Bright dots Bright dots Slightly discolored Discolored Discolored Normal ++ ++ ++ +++ +++ + + ++ – – – – – – – – – – + – – – – + – – – + – – +++ +++ – – – – Results Neuropathology Brain weight was 1639 g in case 1 and 1277 g in case 2, compared to normal (1400 g). The external aspect was normal with no cerebral or cerebellar atrophy. The neuropathological abnormalities were identical in both patients, but more severe in the second case. On sectioning, white matter was affected throughout but to various degrees (Table 1). No atrophy of the cerebrum or corpus callosum was noted. The ventricles were not enlarged. All gray matter structures appeared normal. The most severe lesions were observed in the telencephalic white matter that was grayish, gelatinous and flattened with cavitation restricted to central white matter areas in case 1, but more diffuse in case 2 where only a thin subcortical ribbon of white matter was present (Fig. 1B, C). In these cavitated areas, the white matter resembled a cobweb with sparse and thin myelinated bundles extending from the ventricular wall to the cortical ribbon (Fig. 1C). Myelin was better preserved in U-fibers, internal and external capsules, corpus callosum, anterior commissure and fornix (Fig. 1B, D). Lesions were less severe in infratentorial structures in both cases (Fig. 2A, B, E). Cerebellar white matter was discolored, particularly in central areas like the nucleus dentatus hilus which appeared grayish (Fig. 2E). In the brain stem (Fig. 2A, B), pyramidal and cerebellar tracts were discolored. In both cases the tractus tegmentalis centralis and cerebellar tract decussation in the pons were replaced by two para-median bright white dots (open arrows in Fig. 2A, B). The remaining tracts appeared normal. Histological analysis confirmed the preservation of gray structures (neocortex, basal ganglia and brain stem nuclei), except the cerebellar cortex where Purkinje cells were partially lost. White matter was diffusely involved but to various degrees (Table 1). In discolored white matter areas (i.e. subcortical cerebral and cerebellar white matter, internal and external capsules, cerebral peduncles, pyramidal and cerebellar tracts) myelin fibers were dispersed by vacuoles and their density was decreased (Fig. 1E). In addition, mild astrocytosis was noted. Deeper into cerebellar white matter, these focal swellings of the myelin sheath became more numerous, giving rise to a spongiform, and finally to a pre-cavitated network. Simultaneously, reactive astrocytes were detected and ramified microglia became phagocytic macrophages. In cavitated areas of the telencephalum (Fig. 1D–F), lace-like tissue was nearly devoid of myelin, axons and cells. The rare remaining myelin sheaths, stained with CNP, MBP, PLP and MOG, were mostly located in the perivascular space. They displayed numerous vacuoles that surrounded axons (see Fig. 4I). Most of the axons were naked and were loosely threaded among sparse disseminated reactive astrocytes, phagocytic macrophages and oligodendrocytes (see Fig. 4H). In cavitated areas, large astrocytes with an increased GFAP expression were observed, contrasting with the mild fibrillary astrogliosis in subcortical areas (Fig. 3C, D). The cytoplasm of macrophages was orthochromatic, rarely PAS positive and became sudanophilic, filled with myelin debris, close to cavitated zones (Figs. 3E, F; 4D, F). In the pons, the superior cerebellar peduncles and the tractus tegmentalis centralis were totally devoid of myelin (Fig. 2C). Axons were present, but naked. These areas contained a homogeneous layer of macrophages filled with ORO-positive PAS-negative material (Fig. 2D). Cell numbers were increased (Fig. 3A, B; Table 2) by 200–300% in telencephalic subcortical white matter and 473 Fig. 2A–F Brain stem and cerebellar lesions. A Horizontal section through pons and cerebellar peduncles showing preserved myelin in the median peduncle (arrows) and discolored myelin in superior and inferior cerebellar peduncles (arrowheads). In the dorsal paramedian pons, the superior cerebellar peduncle decussation (open arrow), is unusually bright (case 1). B Horizontal section at the ponto-bulbar junction revealing a bright spot corresponding to the tractus tegmentalis centralis (open arrow) (case 1). C, D Horizontal sections through the pons stained by LFB, and immunostained with anti-PGM1-antibody, respectively, show that the selective loss of myelin in tractus tegmentalis centralis and superior cerebellar peduncle decussation is correlated with macrophage invasion (open arrow). These lesions are specific to the brain stem and to both fiber tracts (case 2). E In the cerebellum, myelin is better preserved, except in nucleus dentatus hilus which appears gray. F Macroscopic section of the cerebellum revealing diffuse white matter discoloration with sparing of subcortical myelin (LFB) (case 2) 474 475 왗 Fig. 3A–F White matter cells. A, B Cell density in cerebral subcortical white matter in the control and in case 1, respectively. C Mild fibrillary astrogliosis in hypercellular areas (case 1). D Same magnification as C showing the increase in GFAP-expression and the large size of astrocytes in cavitated areas (case 2). Note the enhanced staining in the perivascular area and the presence of OROpositive, GFAP-negative cells (arrows). E PGM1-positive microglial cells progressively (top left, subcortical area) become phagocytes close to cavitated areas (bottom right, central area) (case 2). F PGM1-positive cells are distinct from oligodendroglial cells with small, dark, round nuclei. Some are active phagocytes (arrowheads) (case 2) (Cx cortex, ORO Oil Red O, GFAP glial fibrillary acidic protein). A, B Cresyl violet staining; C, D GFAP immunostaining; E, F PGM1 immunostaining. A, B × 6.3; C–E × 10; F × 40 by 40–80% in cerebellar white matter, structures in which myelin was decreased. In both patients, hypercellularity was present in remaining telencephalic white matter but also in cerebellar white matter which was not cavitated. After double immunostaining with anti-GFAP and antiPGM1, only 10–20% of white matter cells were labeled (in equal proportion for both markers) (Fig. 4B), as in the control case (not shown). The remaining unlabelled cells were morphologically homogeneous with small, dark, round nuclei similar to those of oligodendrocytes, and in some cases an abundant and elongated cytoplasm (Fig. 4A, E). Some of these cells were immunolabeled with anti-MBP, -PLP, -MOG (Fig. 4C). Nearly 80–90% expressed CNP within well-developed cytoplasmic expansions (Fig. 4E). All cells with oligodendrocytic morphology expressed CAII (Fig. 4G). Rare MIB1-positive cells were mostly located in deep cerebral white matter, but not in less affected myelinated areas (not shown). MIB1-positive cells were identified as perivascular macrophages and astrocytes by double immunostaining. CNP-positive cells were MIB1 negative (not shown). Macrophages and other glial cells contained no pigment and did not autofluoresce. In semithin sections of cerebellar white matter, numerous oligodendrocytes with visible cytoplasm were intermingled with myelinated axons. In this area, axons were preserved and myelin vacuoles were present (Fig. 5A). Ultrastructurally, oligodendrocytes were identified by chromatin lumps, rough endoplasmic reticulum and lack of intermediate filaments (Fig. 5B). In accordance with light microscopic observations, they were abundant in subcortical cerebral and cerebellar white matter, and had, for the most part, well-developed cytoplasm containing numerous organelles. Myelin sheaths appeared too thin for the diameter of the axons, but periodicity was normal in compacted myelin (Fig. 5E). Previously observed vacuoles corresponded to focal areas of uncompacted myelin close to the axonal membrane (Fig. 5C, D). Underlying axons had normal cytoplasm and a well-organized cytoskeleton. Astrocyte cytoplasm contained dense gliofilaments. The cytoplasmic extensions appeared markedly thick (not shown). In macrophages, membranous debris and lipid droplets could be identified. No lipofuscin deposits were seen. Biochemical and molecular studies During dissection, the cerebellum of the affected brain contained more water than the control. Total protein and lipid contents in the cerebellum were reduced by about 14 and 40%, respectively, in case 1 compared to the control (not shown). Despite severe loss of myelin in the cerebellum (around 50%), the specific activity of CGalT was, on average, about 30% higher than in the control cerebellum (not shown). Loss of polar lipids in the whole cerebellum and myelin (Table 3) was correlated with the loss of myelin. Since an increased number of immature-appearing oligodendrocytes are present in rumpshaker mice [7], myelin mutants with a defective PLP [35], we searched for a similar PLP mutation in case 1. Western blot analysis showed that the amount of PLP was decreased, but its molecular weight was normal (not shown). Southern blot analysis showed a normal restriction pattern of the PLP gene, excluding the presence of a major gene rearrangement. The intensity of enzyme-restricted DNA fragments was identical to the signal observed with an internal Xchromosome-specific probe control (not shown), indicating that the PLP gene was not duplicated in patient 1. The sequence of the coding region of the PLP gene was normal. Discussion Our two patients presented clinical and MRI features compatible with the diagnosis of the CACH syndrome [13, 33, 34, 38]. The neuropathological hallmarks of this new leukodystrophy were unusual. In both cases, cavitating orthochromatic leukodystrophy without atrophy was observed. The intensity of white matter lesions contrasted markedly with the paucity of myelin breakdown products and the relatively mild astroglial and microglial reactions, except within the pons. In this region, numerous macrophages contained myelin debris. Surprisingly, in less-affected white matter areas, oligodendrocytes were abnormally numerous but no significant mitotic activity was detected. There were no neuronal lesions, apart from a loss of Purkinje cells. Axons were relatively well preserved except in cavitated areas. Upon ultrastructural analysis, myelin sheaths appeared thinner than usual. Compaction was normal, but was disrupted in places by vacuoles. Biochemical analysis showed that myelin components were qualitatively normal but several lipids and proteins were reduced. These neuropathological features correlate with the clinical course of the disease. Lesions in the telencephalic white matter displayed features of old lesions, as suggested by the severity of myelin lesions, the paucity of myelin debris and the mild astrocytic and microglial reactivity. These features could be related to the progressive evolution of the disease. Lesions of the pons, however, appeared to be acute and recent, with disappearance of myelin correlated to presence of numerous macrophages containing myelin debris. Bulbar signs appeared late in the disease, preceding death. The loss of Purkinje cells 476 477 Table 2 Cell counts in cases 1 and 2, and in an age-matched control. The results are the mean (SD) of five cell counts in each area Hemispheric white matter (subcortical frontal areas) Cerebellar white matter Case 1 Case 2 Control 1611.3 (52.2) 501.7 (87.1) 1495 (49.5) 636.0 (18.3) 482.2 (83.4) 360.7 (22.0) probably resulted from seizures late in the disease. The cavitated lesions observed on MRI, proton density and flair images were quite similar to those observed macroscopically (Fig. 1A, B). The general histological features of leukodystrophy [30], i.e. exclusive white matter involvement, absence of inflammatory signs, relative axon sparing and thinness of myelin sheaths, were observed in our patients. However, reduced brain weight, ventricle enlargement, fibrillary astrogliosis and loss of oligodendrocytes, all usual in leukodystrophies [30], were missing in our patients. The absence of marked astrogliosis, in spite of severe myelin loss, resulting in cavitated lesions which appeared filled by CSF, was unusual. The most prominent histological feature was the increased density of a homogenous cell population with the morphological features of oligodendrocytes, both in subcortical cerebral white matter surrounding cavitated areas and in the less affected regions such as cerebellar white matter. These cells were also GFAP and PGM1 negative, and CAII positive, confirming their oligodendroglial origin [11]. Some of these oligodendrocytes had unusually abundant cytoplasm. Biochemical analysis showed that CGalT activity was increased. This enzyme is only expressed in oligodendroglial cells, and its expression is developmentally regulated [24]. The increased activity of this enzyme contrasted with the severe loss of myelin lipids and proteins, and might reflect the immaturity or the increased density of the oligodendroglial cells. 왗 Fig. 4A–I Characteristic features of oligodendrocytes and myelin. A Case 1. Cells in subcortical white matter have small, dark, round nuclei. Most of them have a large cytoplasm (arrowheads), H&E staining. B Case 2. Cerebellar white matter double labeled with anti-PGM1 (brown) and anti-GFAP (red). Most of the cells appear unlabeled. C Case 1. The cytoplasm of some oligodendrocytes contains MOG immunoreactivity (arrow). D–F Case 1. Double immunolabeling: CNP (pink) and PGM1 (brown). D Oligodendrocytes (arrowheads) and macrophages (arrow) are easily identifiable. Some macrophages (double arrowhead) have an heterogeneous double-labeled cytoplasm (arrows). E Some of the oligodendrocytes have an hypertrophied, homogeneous CNP-positive cytoplasm. F Some of macrophages have an heterogeneous PGM1- and CNP-positive cytoplasm, due to the presence of myelin debris. G Case 1. All oligodendrocytes have CAII-positive cytoplasm. H Case 2. Lace-like central network containing mostly naked axons (arrowheads). The sparse remaining myelinated fibers contain vacuoles. Note the paucity of cells, LFB-Bodian staining. I Case 2. LFB-Bodian staining shows that vacuoles are in myelin, whereas underlying axons seem preserved (MOG myelin oligodendrocyte glycoprotein, CNP cyclic nucleotide phosphodiesterase, CAII carbonic anhydrase II). A, B, D, H × 40; C, G × 60; E, F, I × 100 Oligodendroglial preservation in leukodystrophies has only been reported in the early stages of Canavan disease [9], and in some adult cases of pigmentary orthochromatic leukodystrophy [12, 27, 37], where brown pigment accumulated in macrophages and glial cells that were autofluorescent, orthochromatic and PAS-positive. Neither of these diagnoses corresponded to our patients. Orthochromatic leukodystrophies with cavitation and an increased density of oligodendrocytes were previously reported in two adults ([10] and case 2 of [39]) and in children ([6] and J-J. Hauw, personal communication), before CACH syndrome was identified. Neuropathological findings and the clinical course of childhood cases suggest that they may have been cases of CACH syndrome. The adult cases raise the possibility that the onset of CACH syndrome may extend into adult life. A primary oligodendrocyte defect was evoked in all cases [6, 10, 39]. An increased number of cells was previously reported in open-brain biopsy specimens from two patients with CACH syndrome [34], but was ascribed to proliferation of astrocytes and microglia. The variability of cell composition in different brain areas may have led to an underestimation of some glial cell populations, particularly when studies were restricted to brain biopsy samples. This is the first report of increased oligodendrocyte density as a hallmark of CACH syndrome. The cause of the increase in oligodendrocyte density, is a matter of speculation. Oligodendroglial proliferation due to remyelination, as in multiple sclerosis [4, 22, 25, 31], can be excluded. Hypercellularity in our cases is not correlated with the severity of myelin defect, and no mitotic activity was detected in oligodendrocytes. An abnormal maturation is suggested by the presence of oligodendrocytes with abundant, elongated and organelle-rich cytoplasm, features that are normal during myelination but which disappear as myelination progresses [21]. An increased number of oligodendrocytes in the myelin mutant rumpshaker mouse has been attributed to abnormal maturation of oligodendrocytes [7, 18, 23], caused by a point mutation in the PLP gene [35]. Molecular analysis has, however, excluded a PLP mutation in patient 1. Finally, the apoptotic process, that normally reduces the number of oligodendrocytes by 50% during the development [1], could have been defective in these patients. Ultrastructural, immunocytochemical and biochemical studies showed that the composition and catabolism of myelin was normal in our patients. However, the myelin sheaths were too thin and, although compaction was normal, it was disrupted in places by vacuoles. The significance of the latter is controversial in postmortem tissue, but they have also been described in brain biopsy specimens [34]. Whether myelin was initially normal in these patients is uncertain. It is clear, however, that demyelination has occurred [38]. It is particularly obvious at the level of the pons. The greater severity of the telencephalic lesions in patient 2 compare to patient 1 suggests a progressive loss of myelin, giving rise to cavitated lesions by a secondary axonal loss. Oligodendrocyte maturation, late steps of myelination, or myelin maintenance may also be 478 Fig. 5 A–E Ultrastructural data in case 2. A On a semithin section of cerebellar white matter, numerous oligodendrocytes with visible cytoplasm are intermingled with myelinated axons (arrowheads). Note axon preservation and myelin vacuoles (open arrows). B Ultrastructural analysis of oligodendrocytes containing abundant cytoplasm, rough endoplasmic reticulum and free ribosomes, but devoid of filaments. C, D Myelin vacuoles appear as focal deformation of the myelin sheath, containing a cytoskeletal-like network, close to the axonal membrane. Note the preserved axonal cytoskeleton. E Myelin sheaths are abnormally thin but periodicity is preserved in compacted areas. A × 100; B, D × 8900; C × 5600; E × 105 000) 479 Table 3 Polar lipid composition in whole cerebellum and in myelin from the cerebellum of case 1 and the age-matched control. The results are the mean (SD) of three separate experiments, expressed as the % of total polar lipids. Other values represent the average of two separate experiments (HFA long chain hydroxy fatty acid, NFA long chain nonhydroxy fatty acid) Sphingomyelin Phosphatidylcholine Phosphatidylserine + inositol Phosphatidylethanolamine + plasmalogen HFA-sulfatides NFA-sulfatides HFA-cerebrosides NHFA-cerebrosides Total phospholipids Total sphingolipids involved. Whether these abnormalities result from a defect intrinsic to oligodendrocytes, abnormal interactions with axons or glial cells, or environmental factors remained to be elucidated [1, 2, 5, 19, 20]. In conclusion, the neuropathological data reported here support the notion that CACH syndrome constitutes a specific entity. Complementary investigations at both morphological, biochemical and molecular level are required for a better comprehension of the physiopathogenesis of this syndrome. Acknowledgments We would like to thank H. Juguelin and J.-L. Nussbaum for their participation in biochemical studies, Dr. A. Tourbah (CHNodes XV-XX, Paris) for the MRI study, A. Chevalier and J. Denni for technical assistance, G. Matteo for preparing the illustrations, and Dr. M. Ruberg for critical review of the manuscript. 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