MLD is a lysosomal lipid storage disorder caused by the deficiency of lysosomal arylsulfatase A (ARSA; EC 3.1.6.8) enzyme (1), or, more rarely, of its activator protein saposin B (SAP-B) (2). The crude birth incidence of the disease ranges from 1/43,000 to 1/70,000. The resulting deficiency of ARSA leads to an accumulation of the sphingolipid cerebroside 3-sulfate (termed sulfatide). This lipid is particularly abundant in the myelin of the nervous system, where it constitutes about 4% of all myelin lipids. Myelin is synthesized by oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS). Functionally, the accumulation of sulfatides affects mostly the nervous system, in particular oligodendrocytes, microglia, and Schwann cells, resulting in severe demyelination of the CNS and PNS. However, sulfatides accumulate also in CNS neurons, contributing to additional neuronal dysfunction and degeneration (3, 4). Storage of sulfatides in gall bladder epithelia and renal tubules results in little or no functional impairment. The term “metachromatic” is based on the observation of a change of the absorbance spectrum (i.e., metachromasia) that is observed when sulfatides bind some types of dyes (such as acid cresyl violet). In the CNS, pathological lesions of MLD include severe myelin loss, astrocytosis, microglia activation, and the presence of macrophages filled with Periodic acid-Schiff (PAS)-positive material. Axonal degeneration is often severe, presumably secondary to myelin loss. Significant abnormalities of motor neurons in the ­spinal cord and neurons in pallidum are present. Peripheral nerves also display severe demyelination with secondary axonal degeneration. Ultrastructural analysis of peripheral nerves show inclusions in Schwann cells related to myelin breakdown. Other inclusions display a periodicity of 5.8 nm and consist of zebra bodies, vacuoles containing irregularly orientated lamellar material and stacks of flattened discs. These inclusions represent the metachromatic sulfatide deposits.

Clinically, signs and symptoms caused by the involvement of CNS and PNS characterize the various forms of MLD. In the majority of the cases, the prognosis is severe, leading to vegetative stage or death within few years after the diagnosis (1). The disease is ­usually classified in four main phenotypes according to the age of onset. The late infantile (LI), which is the most frequent form (approximately 50%) usually manifests in the second year of life. The juvenile variant, with an onset age between 4 and 12 years, is further subdivided into early juvenile (EJ) and late juvenile (LJ), depending on whether the onset is before or after 6 years of age. The term adult MLD refers to patients with onset of neurological symptoms after the age of 12 years. Neurologic symptoms include gait disturbance and clumsiness due to a combination of cerebellar ataxia, pyramidal signs, and/or peripheral nerve involvement of the lower limbs. Inability to walk and stand up follows rapidly with the onset of truncal hypotonia, tetraparesis, dystonia, and athetosis. Cognitive decline occurs together with gait ­disturbances and involves initially mostly visuospatial and executive functions that are often underestimated in young patients. Primary visual loss due to optic nerve atrophy or involvement of occipital white matter occurs at a relative advanced stage. As in many “demyelinating” leukodystrophies, MLD patients frequently develop ­seizures when the disease progresses. While all patients with the infantile form of MLD develop severe peripheral demyelinating neuropathy that can be the first manifestation of the disease for a couple of months, as a rule, the older the patient at onset of symptoms, the less frequent is the peripheral ­demyelinating neuropathy. Adult patients heterozygous for the I179S mutation of the ARSA gene, frequently present with schizophrenia-like behavioral abnormalities, social dysfunction, and mental decline, while motor abnormalities may be scarce (5).

Brain MRI using a 1.5 tesla magnet shows hyposignal in T1 sequence and hypersignal in FLAIR and T2 sequence that affect the white matter in the centrum ovale, the corpus callosum, and the internal capsules (6). Radial stripes having a tigroid aspect are frequently seen, reflecting likely the accumulation of sulfatides in the Virchow spaces (7). Diffusion tensor imaging is a very sensitive biomarker (fractional anisotropy, measurement of apparent diffusion coefficient or ADC) of demyelination and enables the visualization, (tractography with 3D reconstruction) and characterization (measurement of ADC) of demyelinated fasciculi in two and three dimensions (8).

Motor and sensorimotor nerve conductions of the peripheral nerves (lower and upper limbs) are markedly decreased (<20 m/s) in patients who develop demyelinating peripheral neuropathy. As a consequence of peripheral neuropathy but also of spinal cord and brain involvement, somatosensory-evoked potentials (SSEP) from the lower and upper limbs are markedly delayed or ­abolished. Similarly, as a consequence of pyramidal tract involvement from motor cortex to the lower part of the spinal cord, motor evoked responses are also delayed or abolished. Brainstem auditory-evoked potentials (BAEPs) show delayed latencies of I–V interwaves that reflect demyelination in the brain stem. Latency of wave I is ­normal. In contrast, visually evoked responses (P100) can remain normal or nearly normal for years.

ARSA gene mutation allows prediction of the phenotype and the severity of MLD to a relatively good extent, particularly in the late infantile forms of MLD. The genotype–phenotype ­correlation is mostly based upon the distinction of two functionally different types of mutations of the ARSA gene (9): (1) mutations encoding inactive ARSA (0-allele, c.459+1 G>A) and (2) mutations encoding ARSA with residual enzymatic activity (R-alleles). Genotypes comprising two 0-alleles cause the severe late infantile type of MLD. Coincidence of a 0-allele and an R-allele induces predominantly the intermediate juvenile type, whereas two R-alleles usually cause the milder adult type of MLD. More than 120 ARSA mutations have been described to date according to the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php). The null allele c.459+1 G>A and the Pro426Leu alleles (usually observed in adult MLD patients presenting with motor symptoms) account respectively for 25% and 18.6% of MLD alleles (3, 9). Most other mutant alleles have been found in single families (10), but there is now an international effort to systematically search for disease-causing mutation in MLD and correlate these ARSA gene mutations, as well as polymorphisms in the normal ARSA gene, with the phenotype.

ARSA-deficient mice lack ARSA activity and develop a disease that resembles MLD, but which is much less severe and, in particular, does not include cerebral demyelination (11). To establish ARSA-deficient mice, murine embryonic stem cells with a null mutation of the ARSA gene were injected into C57BL/6 blastocysts and chimeric male mice were bred with 129/OlaHsd female mice to generate ARSA+/– mice, and subsequently ARSA–/– mice with a pure 129/OlaHsd background.

ARSA-deficient mice show an age-dependent increase in brain sulfatides from the age of 3 months that can be assessed using thin-layer chromatography (11, 12, 13). Measurement of sulfatide isoforms by tandem mass spectrometry allows to identify specific neuronal (C18:0 isoform) and oligodendrocyte (C24:0) species of this lipid. Oligodendrocyte sulfatide species accumulate at higher levels than neuronal sulfatide species (Aubourg et al., unpublished). There is an age-related increase in C24:0 species and a corresponding decrease in C16:0–C20:0 species. Histo­logically, sulfatide storage can be assessed using Alcian blue (11, 12, 13) staining and starts to be obvious at 9 months of age, mostly in the white matter (corpus callosum, hippocampal fimbria, internal capsule, and optic nerve). On light microscopical level, sulfatide storage in the white matter results in two different morphologies (11, 14): (1) in the form of numerous fine granules arranged immediately adjacent to myelinated nerve fibers that correspond likely to accumulation of sulfatides in oligodendrocytes, and (2) as clusters of larger granules within swollen cells interspersed within the white matter that correspond to astocytes and microglia.

Despite significant accumulation of sulfatides in oligodendrocytes and obvious sulfatide storage in the white matter, ARSA-deficient mice do not develop any sign of cerebral or cerebellar demyelination, even at 18 months of age, when assessed by immunostaining with anti-proteolipid protein (PLP), anti-myelin basic protein (MBP), anti-2,3-cyclic nucleotide 3-phosphodiesterase (CNPAse) antibodies or Luxol fast blue staining. There is however some delay in myelination (15). At 2 weeks of age, ARSA-deficient mice show a substantial reduction in MBP mRNA and protein. This is confirmed by immunohistochemical analysis. MBP mRNA and protein, however, reach normal levels at 3 weeks of age. PLP and myelin and lymphocyte protein (MAL) mRNA are also reduced in ARSA-deficient mice at 2 weeks of age, whereas the level of PLP mRNA is normal at 26 weeks of age. In situ hybridization reveals no significant changes in the number of myelinating oligodendrocytes or oligodendrocyte precursor cells. This suggests that oligodendrocyte differentiation is normal. The excess of sulfatide does not seem to affect the survival of normal neural stem cells, at least when these cells are transplanted in the brain of ARSA-deficient mice (16). However, it might affect the differentiation of normal neural stem cells in mature neurons or oligodendrocytes (16).

Neuronal storage of sulfatides is also observed in several layers of cerebral cortex, nuclei of brain stem, diencephalon, spinal cord, and cerebellum (12, 13). Fluoro-Jade B specifically stains degenerating neural cells. Brain sections from 18-month-old untreated MLD mice show few Fluoro-Jade B-positive cells scattered throughout the cerebral and cerebellar cortex, and the pons.

Immunostaining with lectin or Iba1 antibody reveals an increased number of swollen amoeboid cells that characterize activated microglia from the age of 12 months. Similarly, marked astrogliosis is observed progressively from the age of 12 months, mostly in the white matter (corpus callosum, fimbria, cerebellar white matter), hippocampus, and pons of ARSA-deficient mice.

Periodic acid-Schiff (PAS)-reactive material reflecting lipid storage is detected from the age of 12 months, mainly in the cytoplasm of large swollen macrophages within the white matter.

ARSA-deficient mice display progressive loss of their Purkinje cells starting after the age of 14–16 months (12, 16, 17). Examination of cerebellar histology shows that 2-year-old ARSA-deficient mice have lost most of the calbindin immunoreactivity from their Purkinje cell dendrites and show simplified dendritic architecture. Recordings of unitary potentials and stimulation of climbing fibers on cerebellar slices from 2-year-old mice indicate that although the main cerebellar synapses seem to be present and functioning physiologically, the climbing fibers of ARSA-deficient mice may have enhanced effects on Purkinje cell activity (17).

Neuronal damage is dramatic in the inner ear of ARSA-deficient mice (11, 18). Already at 8 months of age, the number of acoustic ganglion cells, as well as their corresponding myelinated nerve fibers, are greatly reduced. Remaining ganglion cells are surrounded by Schwann cells containing large amounts of storage material. In younger mice (6 months), the number of acoustic ganglion cells appears normal, but the neurons and the surrounding Schwann cells show marked sulfatide storage. Sulfatide storage is also observed in the vestibular ganglion, but without reduction of ­neurons and nerve fibers. A decreased number of neurons is also observed in the ­ventral cochlear (after 8 months) and trapezoid nucleus (after 20 months).

As a consequence of the inner ear lesions, the amplitude of waves recorded during the study of BAEPs is severely decreased in 6-month-old ARSA-deficient mice (12, 19). The latency of the I–V interpeak is no longer reliably measurable at 6 months, and BAEPs are completely abolished at 9 months of age with a complete disappearance of wave I.

In the peripheral nerves, sulfatide storage is also detected in Schwann cells, but there are no signs of demyelination or axonal damage. On a different but not well-defined background, one group reported that ARSA-deficient mice exhibit reduced nerve conduction velocity, without however significant lesions of myelinated axons (20). These electrophysiological abnormalities have not been observed on the pure 129/OlaHsd background (De Deyn P and Aubourg P, unpublished).

The gall bladder, intrahepatic bile ducts, exocrine pancreatic ducts, respiratory epithelium, and, to a lesser extent, testicular Sertoli cells show sulfatide storage (21). Hepatocytes, pancreatic islets, adrenal glands, and gastric epithelium are unaffected. Apart from some differences, the topographic distribution of the sulfatide storage resembles that of human MLD.

Most distinct behavioral abnormalities only appear beyond 1 year of age, and include impaired motor coordination on the rotarod, abnormal gait, and deafness (11–13, 17, 19, 22). Older mice are generally more active and display a stronger grip. In the open-field, ARSA-deficient mice make less corner entries, and older animals display an increased path length. During the training phase of the passive avoidance task, ARSA-deficient mice show longer step-through latencies, but with better results during the testing phase than the training, indicating successful learning, clearly in contrast with the cognitive decline observed in patients.

In respect to cerebellar ataxia that is observed early in nearly all MLD patients, overt signs of ataxia are not observed in 6-month-old ARSA-deficient mice. Velocity, time and distance moved, and number of ambulatory episodes are similar to normal mice with the same genetic background.

Quantitative gait analysis using video-tracking during open-field exploration reveals however that ARSA-deficient mice ­display increased hind base width and increased stride length for all paws at 6 months (23). Their covert motor incoordination is evident in a correlation analysis which unveiled decreased harmonization of concurrent gait parameters. Furthermore, various behavioral observations indicate emotional alterations, including reduced proactive anxiety and anhedonia. ARSA-deficient mice spend more time in the open arms of the elevated plus maze, make less corner entries in the open-field test, and display longer step-through latencies during the training phase of the passive avoidance test. Additionally, 6-month-old ARSA-deficient mice show lower response rates in scheduled appetitive conditioning. Six-month-old mice also show decreased response rates in scheduled operant responding that is likely more the consequence of emotional dullness or inattentiveness rather than due to neuromotor defects (23). In conclusion, several data sets confirm the lack of pronounced cognitive deficits in ARSA-deficient mice below the age of 1 year.

One reason for this discrepancy between the human and mouse phenotype could be that sulfatides accumulate to a much smaller extent (sixfold less) in the brain of ARSA-deficient mice compared to the brain of MLD patients (24). Despite the fact that both at the biochemical and histological level, sulfatides accumulate mostly in oligodendrocytes and white matter, it is generally considered that the motor and behavioral abnormalities observed in MLD mice reflect neuronal dysfunction. In fact, it is likely that the motor and behavioral abnormalities observed in MLD mice are the consequences of axonal dysfunction due to primary oligodendrocyte impairment. It is indeed clearly established that once myelination has been achieved, oligodendrocytes serve two functions: (1) to preserve axons, and (2) to maintain myelin integrity. Oligodendrocyte dysfunction can result in axonal dysfunction without demyelination (25, 26). There is also evidence that sulfatides play an important role at the nodes of Ranvier, i.e., at the axonal–glial junction (see below).

ARSA-deficient mice with increased synthesis of sulfatides in neurons or oligodendrocytes and Schwann cells have been ­generated. The ARSA-deficient mice overexpressing the sulfatide-synthesizing enzymes UDP-galactose:ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST) in neurons show a more marked neuronal sulfatide storage than the ARSA-knockout mouse (27). Transgenic CGT/ARSA(–/–) mice develop more severe neuromotor coordination deficits and weakness of hind- and forelimbs. Light and electron microscopical analyses demonstrate nerve fiber degeneration in their spinal cord, which is well correlated with higher amounts of Alcian blue-positive material. The difference between transgenic and nontransgenic ARSA mice is even more pronounced in the brain. The motor neurons of the facial and hypoglossal nuclei display relatively intense sulfatide storage, which is not a feature of ARSA-deficient mice. As in the forebrain, sulfatide storage is also more prominent in the isocortical lamina 5, CA1 and CA3 regions of the hippocampus, and in the thalamus and amygdaloid nucleus. The area postrema, dorsal vagal nucleus, nucleus of solitary tract, the inferior olive, cerebellar Purkinje cells, and the caudate putamen show however no sulfatide storage. Surprisingly, transgenic CGT/ARSA(–/–) mice do not develop degeneration and loss of Purkinje cells, at least at 6 months of age. Despite the increased accumulation of sulfatides in neurons, neuronal apoptosis cannot be detected using the TUNEL assay. Although already present in ARSA-deficient mice, more significant cortical hyperexcitability, with recurrent spontaneous cortical EEG discharges lasting 5–15 s is observed in transgenic CGT/ASA(–/–) mice (27).

A different strategy was used to generate ARSA-deficient mice with increased synthesis of sulfatides in oligodendrocytes and Schwann cells. Sulfatides are synthesized in the Golgi apparatus by galactose-3-O-sulfotransferase-1 (Gal3st1) transferring sulfate from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to galactosylceramide (GalC). The overexpression of the Gal3st1 gene under control of the PLP promoter in oligodendrocytes and Schwann cells of ARSA-deficient mice leads also to a significant, more marked increase of sulfatide storage in the nervous system than in simple ARSA-knockout mice (28). These transgenic mice develop myelin pathology in the CNS and particularly in PNS. Until 1 year of age, appearance and behavior of transgenic Gal3st1 ARSA-deficient mice are indistinguishable from nontransgenic ARSA-deficient mice. Older transgenic mice, however, develop behavioral abnormalities not seen in ARSA-deficient mice. When suspended by their tails, transgenic Gal3st1 ARSA-deficient mice often grasp their hind limbs to their body. Later in life, they develop a progressive hind-limb paralysis absent in simple ARSA-deficient mice. Sulfatide storage is clearly more marked in spinal cord and peripheral nerves, as revealed by Alcian blue staining. In the peripheral nerves, Schwann cells are filled with large amount of sulfatides and myelin thickness is reduced. The number of Schwann cells is increased, but there is no absolute evidence of a demyelination/remyelination process. In contrast to ARSA-deficient mice, nerve conduction velocities and compound muscle action potentials (CMAPs) are clearly reduced, while F-wave latency is significantly increased. While the number of lipid-filled macrophages is increased in the optic nerves and corpus callosum, there is however no clear evidence of marked demyelination in the cerebral white matter. One has to rely upon electronic microscopy to demonstrate that the thickness of the myelin sheaths is inhomogeneous in the corpus callosum and that some axons are hypo- or unmyelinated. Thus, the major effect of an increased synthesis of sulfatides in myelin-forming cells is observed in the peripheral nerves and spinal cord, not in the brain. Brain MRI with a 5 or 7 Tesla magnet has not yet been performed to determine if myelin abnormalities can be detected in the brain of transgenic Gal3st1 ARSA-deficient mice. Whether the lack of obvious cerebral demyelination results from the relative weakness of the PLP promoter or from other factors is not known.

In the perspective of evaluating enzyme-replacement therapy (ERT), the ARSA-deficient mice has been “humanized.” A cysteine-to-serine substitution was introduced into the active site of the human ARSA gene and the resulting inactive Cystein69Serine variant was constitutively expressed in ARSA-deficient mice. This mouse model of MLD is therefore tolerant to the repeated injection of recombinant human ARSA enzyme (29). The phenotype of this new ARSA-deficient mouse has not been described in detail, but seems similar to the knockout ARSA mice (30). In the same line, for evaluation of ERT, a PLP-CST/hASA-c69s/ASA(–/–) mouse has recently been generated. This double transgenic ARSA-deficient mouse expresses the CST enzyme in oligodendrocytes under the control of the PLP promoter and the pathogenic Cystein69Serine mutation of the human ARSA gene.

The physiopathology of MLD is still not fully understood, but significant progress has been made in this field (4, 31). Together with their precursor galactosylceramide (GalCer), sulfatides account for almost one third of myelin lipids and are exclusively found on the extracellular leaflet of the membranes. As all sphingolipids, sulfatides exhibit variation of their structure due to different acyl chain lengths, which can also be 2-hydroxylated. Because neurons synthesize especially ceramides containing C18:0-fatty acids, neuronal sulfatides are enriched in C18:0-species. In contrast, oligodendrocytes are enriched in C22:0/C26:0 sulfatide species. Sulfatides do not seem to have the same topology on oligodendrocytes and neurons. In the myelin sheath (an expansion of oligodendrocyte membrane) the head group of sulfatides faces outwards from the cell. In neurons (and astrocytes), they are localized in intracellular compartments. It is therefore likely that accumulation of sulfatides does not have the same pathogenic effects in oligodendrocytes and neurons.

Although sulfatides are mainly found in oligodendrocytes and Schwann cells, they are also present in neurons and astrocytes. However, it is not clear whether sulfatides are synthesized by ­neurons or astrocytes themselves or imported, i.e., via lipoprotein endocytosis, particularly apolipoprotein E-containing ­lipoproteins secreted by astrocytes.

During oligodendrocyte differentiation, sulfatides are first detected at the stage of immature oligodendrocytes, and their synthesis is upregulated before oligodendrocytes wrap myelin around axons. This suggests that sulfatides may not only fulfill a role as a structural component of myelin. The initiation of myelination appears to be stimulated by sulfatides, at least in cultured Schwann cells. Sulfatides bind to components of the extracellular matrix, like tenascin-R or laminin, and can generate signaling via the c-src/fyn kinase pathway. However, decreased synthesis or degradation of sulfatides does not seem to significantly affect myelination and oligodendrocyte survival both in mouse and human, but rather myelin maintenance.

Sulfatides produced by oligodendrocytes likely have an important role in axonal maintenance. As discussed above, most of behavioral and motor abnormalities observed in ARSA-deficient mice are likely the consequence of axonal dysfunction. The role of sulfatides in axonal function is exemplified by a reduced axon caliber and extended axonal protrusions at the nodes of Ranvier in adult CST-deficient mice. In addition, these nodes contain abnormal, enlarged vesicles and an unusual absence of contactin-associated protein (Caspr) and NF155 clusters. As a component of detergent-resistant myelin membranes (lipid rafts), sulfatides could be involved in recruiting proteins to the myelin but also to the axonal membrane. At 18 months of age, ARSA-deficient mice display a 35% decrease in the GalCer content of the brain (12 32). This decrease could reflect instability of the myelin sheaths and/or result from an increase in the degradation of GalCer (15). The sulfatide/GalCer ratio increases however progressively as ARSA-deficient mice become older (12, 13). The conservation of this ratio might be required to form and to recruit NF155 into stable lipid rafts at axon–glial junctions. NF155 clusters are essential to concentrate Caspr and contactin in the axonal membrane, thereby forming stable axon–glial junctions.

MAL is mistargeted in ARSA-deficient mice (24). In view of its binding to sulfatides and the sulfatide-dependent missorting of MAL in sulfatide-storing kidney cells, MAL-deficiency might also impair transport of sulfatides to “paranodal lipid rafts,” causing destabilization of the axon–glial junction.

Sulfatides accumulate in neurons of ARSA-deficient mice. However, loss of neurons does not always correlate with sulfatide storage, as for example cerebellar Purkinje cells degenerate in old ARSA -deficient mice without any indication for lipid accumulation. This raises the important question: To what extent is intracellular lysosomal storage or elevated sulfatide levels in the plasma membrane involved in the pathogenesis of the disease? Secondary changes in lipids that have been observed in ARSA-deficient mice, as in other lysosomal storage disorders, include increase in ­gangliosides GM2 and GD3 (13) and reduced cholesterol levels in old ARSA-deficient mice (33). Although GM2 accumulation in ARSA-deficient mice is only moderate, it might also affect neuronal function. The slight (15%) reduction in cholesterol level observed in ARSA-deficient mice might however be relevant for the pathogenesis of the disease, given the role of this lipid in signal transduction, particularly in lipid rafts.

Neuronal hyperexcitability in ARSA-deficient mice suggests modulation of electrophysiological properties by sulfatides. This might be relevant for the pathogenesis of MLD. Sulfatides potentially could affect functional properties of ion channels, ion pumps, receptors, or transporters. Sulfatides are thought to modulate fish gill Na⁺/K⁺-ATPase activity, though it is not known if this applies also to mammalian Na⁺/K⁺-ATPase.

In vitro, excess of sulfatides changes the morphology of primary microglia to their activated form, and it induces the production of various inflammatory mediators in primary microglia and astrocytes (34). Moreover, sulfatides rapidly trigger the phosphorylation of p38, ERK, and JNK, and they markedly enhance the NF-kappaB and AP1-binding elements. Sulfatide-triggered inflammatory events appear to occur at least in part through an L-selectin-dependent mechanism. L-selectin is dramatically downregulated upon exposure to sulfatide, and inhibition of L-selectin results in suppression of sulfatide-triggered responses. Thus, sulfatide excess may induce and exacerbate the inflammatory response that probably plays a role in the death of neurons and oligodendrocytes.