Search

GM1 gangliosidosis is a storage disorder with autosomal recessive inheritance caused by deficiency of β-galactosidase (GLB1), which is a lysosomal hydrolase, due to mutations in GLB1. We describe here an autopsy case of GM1 gangliosidosis in a female patient who survived for 38 years with a long period of artificial respiratory support (ARS). She was born after a normal pregnancy and delivery. Although development was normal until one year old, she was unable to walk at two years old and started having seizures by nine years old. At 21 years old, she became unable to communicate and was bed-ridden. At 36 years old, she suffered from pneumonia and required ARS. She died of pneumonia at 40 years old. Neuropathological examination revealed severe atrophy, predominantly found in the frontal lobes. Microscopically, severe gliosis and neuronal loss were observed in the cerebral cortex, putamen, cerebellum, the latter including Purkinje cell and granule cell layers. The hippocampus was relatively preserved. Severe neuronal swelling was observed in the limbic regions and stored a material in these neurons negative for periodic acid-Schiff (PAS). A PAS-positive granular storage material in neurons and macrophages was mainly observed in the brainstem and limbic regions. Exome analysis showed a known c.152T>C (p.I51T) variant that has been described in type III patients and a novel c.1348-2A>G variant in GLB1. Detailed analysis of reverse transcription-polymerase chain reaction products of GLB1 mRNA revealed that these variants were present in a compound heterozygous state. In our case, clinical features and neuropathological findings were most consistent with type II, although the entire course was longer than any previously reported cases. This may be explained by the residual enzyme activity in this patient whose severity lay between types II and III. Our finding of relative preservation of the limbic regions suggests that neuronal loss in GM1 gangliosidosis has regional selectivity.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is caused by ABCD1 mutations. A cerebello-brainstem dominant form that mainly involves the cerebellum and brainstem is summarized in a review of the literature, with autopsy confirmed cases exceedingly rare. We report a 69-year-old white man who was diagnosed with this rare disorder and describe neuropathologic, ultrastructural and genetic analyses. He did not have adrenal insufficiency or a family history of X-ALD or Addison’s disease. His initial symptom was temporary loss of eyesight at age 34 years. His major symptoms were chronic and progressive gait disorder, weakness in his lower extremities, and spasticity, as well as autonomic failure and cerebellar ataxia suggesting possible multiple system atrophy (MSA). He also had seizures, hearing loss, and sensory disturbances. His brain MRI showed no obvious atrophy or significant white matter pathology in cerebrum, brainstem or cerebellum. He died at age 69 years with a diagnosis of multiple system atrophy. Microscopic analysis showed mild, patchy myelin rarefaction with perivascular clusters of PAS-positive, CD68-positive macrophages in the white matter most prominent in the cerebellum and occipital lobe, but also affecting optic tract and internal capsule. Electron microscopy of cerebellar white matter showed cleft-like trilamellar cytoplasmic inclusions in macrophages typical of X-ALD, which prompted genetic analysis that revealed a novel ABCD1 mutation, p.R163G. Given the relatively mild pathological findings and long disease duration, it is likely that the observed pathology was the result of a slow and indolent disease process. We described a patient who had sporadic cerebello-brainstem dominant form of X-ALD with long clinical course, mild pathological findings, and an ABCD1 p.R163G substitution. We also review a total of 34 cases of adult-onset cerebello-brainstem dominant form of X-ALD. Although rare, X-ALD should be considered in the differential diagnosis of MSA.

The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessively inherited disorders collectively considered to be one among the most common pediatric neurodegenerative lysosomal storage diseases. Four main clinical subtypes have been described based on the age at presentation: infantile, late infantile, juvenile and adult types. In addition, rare congenital cases of NCL have been reported in the literature. Previously, a homozygous mutation in the cathepsin D gene has been shown to cause congenital NCL in a patient of Pakistani origin. We report a case of a 39-week estimated gestational age female infant with severe microcephaly and hypertonia, whereas MRI showed generalized hypoplasia of the cerebral and cerebellar hemispheres. The infant died on day two after birth. Postmortem examination revealed a small, firm brain with extensive neuronal loss and gliosis. Remaining neurons, astrocytes and macrophages contained PAS-positive storage material with granular ultrastructure and immunoreactivity against sphingolipid activator protein D. A diagnosis of congenital NCL was rendered with a novel mutation, c.299C > T (p.Ser100Phe) in exon 3 of the cathepsin D gene. In the patient fibroblasts, cathepsin D activity was marginal, but the protein appeared stable and normally processed. This was confirmed in overexpression studies. Importantly, by identification of the mutation in the family, we were able to confirm the first prenatal diagnosis excluding cathepsin D deficiency in the younger sibling of the patient.

Globoid cell leukodystrophy (GLD; Krabbe's disease), caused by a genetic galactosylceramidase deficiency, affects both the central and peripheral nervous systems (CNS and PNS). Allogenic hematopoietic stem-cell transplantation (HSCT) has been beneficial for clinical improvement of this disease. However, recent reports by Siddiqi et al. suggested that none of their transplanted patients achieved complete normalization of their peripheral nerve function, despite the well-documented remyelination of the CNS and PNS in the treated patients. We hypothesized that the PNS dysfunction in GLD is due to altered Schwann cell-axon interactions, resulting in structural abnormalities of the node of Ranvier and aberrant expression of ion channels caused by demyelination and that the persistence of this altered interaction is responsible for the dysfunction of the PNS after HSCT. Since there has not been any investigation of the Schwann cell-axonal relationship in twitcher mice, an authentic model of GLD, we first investigated structural abnormalities, focusing on the node of Ranvier in untreated twitcher mice, and compared the results with those obtained after receiving bone marrow transplantation (BMT). As expected, we found numerous supernumerary Schwann cells that formed structurally abnormal nodes of Ranvier. Similar findings, though at somewhat variable extent, were detected in mice treated with BMT. Activated supernumerary Schwann cells expressed GFAP immunoreactivity and generated Alcian blue-positive extracellular matrix (ECM) in the endoneurial space. The processes of these supernumerary Schwann cells often covered and obliterated the nodal regions. Furthermore, the distribution of Na(+) channel immunoreactivity was diffuse without the concentration at the nodes of Ranvier as seen in wild-type mice. Neither K(+) channels nor Neurexin IV/ Caspr/ Paranoidin (NCP-1) were detected in the twi/twi sciatic nerve. The results of our study suggest the importance of normalization of the Schwann cell-axon relationship for the functional recovery of peripheral nerves, when one considers therapeutic strategies for PNS pathology in GLD.

A 44-year-old Japanese man with elevated growth hormone levels and gradual deterioration of mental and renal function was admitted to the hospital. With his deteriorated general condition and renal failure, the patient developed pulmonary thromboembolism and died of respiratory failure. Autopsy examination was conducted, which revealed abnormal accumulation or intracytoplasmic storage of lipid-rich material in the small blood vessels, kidney, heart, and nervous system. After postmortem pathologic studies, including light-microscopic histochemistry, electron microscopy, and biochemical analysis of the stored lipid contents, a final diagnosis of Fabry disease was made. Histopathologic examination revealed a unique vasculopathy characterized by the presence of abnormal intracytoplasmic lipid inclusions and vascular remodeling. With regard to the clinical presentation of acromegaly, hyperplasia but not adenomatous transformation of the acidophils of the anterior pituitary gland with immunohistochemical detection of growth hormone within the cells was noted. In this case, the complication of acromegaly with hyperplasia of the acidophilic cells of the anterior pituitary gland and the unique vasculopathy causing significant organ failure, mainly of the kidney, heart, and central nervous systems, possibly as a result of microcirculatory failure, are considered to be not incidental findings but to be intimately involved in the pathogenesis of Farby disease.

Varieties of neuropathological disorders are caused by a perturbation of normal developmental processes, resulting from insults by heterogeneous etiologic factors. These factors trigger the sequence of molecular, biochemical, and morphologic alterations of the brain, resulting morphologically and/or functionally abnormal brain. The resulting brain contains basic components of the normal brain but is assembled in an abnormal way. The developmental stage when the insults occur appears to largely dictate the outcome of the pathological processes. Depending on the developmental stage involved, the morphology of the brain may be grossly abnormal or is apparently normal but functionally abnormal. The brain development progresses in an orderly fashion and can be divided into several major developmental stages; the neurulation (neural tube formation), ventral induction (formation of prosencephalon), neuroepithelial cell proliferation and migration, neuroglial differentiation and establishment of neuronal circuits. The perturbation of these developmental stages results in uniquely specific pathological outcome, regardless of the etiologic factors/agents. In this review, I will briefly discuss the normal pattern of brain development and neuropathology of the representative disorders resulting from the deviation of normal developmental processes in the individual developmental stage.

Spontaneously occurring genetic lysosomal storage diseases are as rare in other mammalian species as in man. However, the advent of gene targeting technology has revolutionized the state of animal models of genetic diseases. Nearly all lysosomal storage diseases known in man have been duplicated in the mouse. The technology now allows, not only complete inactivation of endogenous genes, but also the introduction of essentially any type of mutation. These animal models can overcome many of the limitations inherent in studies of human patients - rarity of the disease, extremely complex genetic background and logistical and ethical constraints in the design and execution of experiments with human subjects. For example, genetic manipulations of germ cells or cross-breeding experiments between two mutants are readily feasible with animal models. Two major areas of the utility of animal models are the clarification of the pathophysiology/ pathogenetic mechanism of disease and the exploration of therapeutic approaches. Examples of experiments using animal models of lysosomal storage disease are presented, primarily from studies undertaken in our own laboratory. Conclusion: Animal models have proved invaluable in extending our knowledge of the lysosomal storage diseases and exploring potential therapies.

We observed severe ataxia in mice homozygous for modification of the Pparg locus. Genetic analysis and nucleotide sequencing revealed that ataxia is caused by a T692K substitution in plasma membrane calcium ATPase 2 (Pmca2), which is tightly linked to Pparg, but not by modified PPARgamma itself. We traced this mutation and found that it arose spontaneously during clonal expansion of the targeted embryonic stem (ES) cells. Consistent with the deafwaddler phenotype in other Pmca2 mutants, homozygous T692K Pmca2 mutants exhibit severe balance disorder, impaired neurologic reflexes, and motor coordination, and have profound hearing loss. Heterozygous mutants have normal movement and motor function but are severely deficient in hearing. Our findings represent a cautionary example since, although rare, spontaneous mutations do arise in ES cells during culture and hitchhike onto the targeted gene mutation.

Prostaglandin (PG) D2is well known as a mediator of inflammation. Hematopoietic PGD synthase (HPGDS) is responsible for the production of PGD2involved in inflammatory responses. Microglial activation and astrogliosis are commonly observed during neuroinflammation, including that which occurs during demyelination. Using the genetic demyelination mousetwitcher, a model of human Krabbe’s disease, we discovered that activated microglia expressed HPGDS and activated astrocytes expressed the DP1receptor for PGD2in the brain of these mice. Cultured microglia actively produced PGD2by the action of HPGDS. Cultured astrocytes expressed two types of PGD2receptor, DP1and DP2, and showed enhanced GFAP production after stimulation of either receptor with its respective agonist. These results suggest that PGD2plays an important role in microglia/astrocyte interaction. We demonstrated that the blockade of the HPGDS/PGD2/DP signaling pathway using HPGDS- or DP1-nulltwitchermice, andtwitchermice treated with an HPGDS inhibitor, HQL-79 (4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine), resulted in remarkable suppression of astrogliosis and demyelination, as well as a reduction in twitching and spasticity. Furthermore, we found that the degree of oligodendroglial apoptosis was also reduced in HPGDS-null and HQL-79-treatedtwitchermice. These results suggest that PGD2is the key neuroinflammatory molecule that heightens the pathological response to demyelination intwitchermice.

Genetically authentic animal models of human lysosomal diseases occur spontaneously in many mammalian species. However, most are among larger domestic or farm animals with only two well‐defined genetic lysosomal diseases known among rodents. This status changed dramatically in recent years with the advent of the combined homologous recombination and embryonic stem cell technology, which allows directed generation of mouse models that are genetically equivalent to human diseases. Almost all known human sphingolipidoses, two mucopolysaccharidoses and aspartylglycosamin‐uria have so far been duplicated in mice and more are expected in the near future. This technology also allows generation of mouse mutants that are not known or are highly unlikely to exist in humans, such as “double‐knockouts.” These animal models will play an important role in studies of the pathogenesis and treatment of these disorders. While the utility of these mouse models is obvious, species differences in brain development and metabolic pathways must be always remembered, if the ultimate goal of the study is application to human patients.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dually functional protein, acting both as a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. L-PGDS is expressed in oligodendrocytes (OLs) in the central nervous system and is up-regulated in OLs of the twitcher mouse, a model of globoid cell leukodystrophy (Krabbe's disease). We investigated whether up-regulation of L-PGDS is either unique to Krabbe's disease or is a more generalized phenomenon in lysosomal storage disorders (LSDs), using LSD mouse models of Tay–Sachs disease, Sandhoff disease, GM1 gangliosidosis and Niemann–Pick type C1 disease. Quantitative RT-PCR revealed that L-PGDS mRNA was up-regulated in the brains of all these mouse models. In addition, strong L-PGDS immunoreactivity was observed in OLs, but not in either astrocytes or microglia in these models. Thus, up-regulation of L-PGDS appears to be a common response of OLs in LSDs. Moreover, surface plasmon resonance analyses revealed that L-PGDS binds GM1 and GM2 gangliosides, accumulated in neurons in the course of LSD, with high affinities (KD = 65 and 210 nm, respectively). This suggests that L-PGDS may play a role in scavenging harmful lipophilic substrates in LSD.

Krabbe disease is a genetic demyelinating disease caused by a deficiency of galactosylceramidase. The majority of cases are of infantile onset with rapid clinical course. A rare late onset form with milder clinical symptoms also exists. The latter form has been reported to respond well to the bone marrow transplantation (BMT) therapy. We tested whether the BMT could be an effective therapy for the mouse model of the late onset form, saposin-A-/- (SAP-A-/-) mice. We used green fluorescent protein transgenic mice as the donors. Chimeric SAP-A-/- mice that received BMT showed very little evidence of neurologic symptoms. At postnatal day 190 when severe demyelination was evident in naive SAP-A-/- mice, demyelination was virtually absent in the brain of chimeric SAP-A-/- mice. Presence of residual enzyme activity, at the time of rapid myelination in SAP-A-/- mice, appears to limit initial inflammatory responses and macrophage infiltration, thereby preventing progression of demyelination in the CNS in SAP-A-/- mice. In contrast, the peripheral nerves showed features of hypertrophic neuropathy with hypomyelination and onion bulb formation, suggesting that there are different cellular responses to the BMT in the CNS and PNS.

Niemann-Pick disease type C (NP-C) is an autosomal recessive neurovisceral storage disease with neurodegeneration caused by mutations in either the NPC-1 or NPC-2 gene. The murine ortholog of NPC-1 is mutated in BALB/c npc(nih) and this mutant mouse shows equally conspicuous neurodegeneration and loss of neurons. However, the molecular mechanisms causing neurodegeneration in NP-C remain elusive. Here, we report the presence of apoptotic cells detected by both TUNEL staining and electron microscopy in the cerebrum and cerebellum of human patients and the mouse model. Moreover, we found that with progression of the disease process leading to neuronal cell death, an up-regulation of genes involved in the TNF-alpha death pathway caspase-8, FADD, TNFRp55, TRADD, and RIP-by an RNA protection assay. Furthermore, RT-PCR showed that TNF-alpha mRNA expression level also increased up to 30-50-fold in the cerebellum of 7- and 9-week-old NP-C mice compared with wild-type mice. Elevated expression of TNF-alpha was detected in both neurons and astrocytes with TNF-alpha-expressing astrocytes distributed in the affected brain regions. Collectively, our results suggest that the cell death in the brain of NP-C disease occurs through apoptosis and it is mediated by the TNF receptor superfamily pathway.

The twitcher mouse is an authentic murine model of a genetic demyelinating disease globoid cell leukodystrophy. Allogeneic bone marrow transplantation (BMT) in twitcher mice resulted in the clinicopathological improvement. Thus, using green fluorescent protein (GFP) transgenic mice as the donor, we investigated the behavior and fate of the donor cells and the possibility of transdifferentiation of the donor cells into neuroglial cells in the chimeric twitcher mice. GFP(+) cells were found throughout the brain, most conspicuous in the areas of demyelination. The donor GFP(+) cells expressed RCA-1, a marker for microglia/macrophages but were never exceed 70% of the entire population of the RCA-1(+) microglia/macrophages. There was no convincing evidence that GFP(+) donor cells expressed markers for neurons, astrocytes, or oligodendrocytes. We concluded BMT is therapeutic for this model. However, this effect is not mediated by donor cells transdifferentiation in the brain.

To understand mechanisms that may underlie the progression of a demyelinated lesion to a chronic state, we have used the cuprizone model of chronic demyelination. In this study, we investigated the fate of oligodendrocytes during the progression of a demyelinating lesion to a chronic state and determined whether transplanted adult oligodendrocyte progenitors could remyelinate the chronically demyelinated axons. Although there is rapid regeneration of the oligodendrocyte population following an acute lesion, most of these newly regenerated cells undergo apoptosis if mice remain on a cuprizone diet. Furthermore, the oligodendrocyte progenitors also become progressively depleted within the lesion, which appears to contribute to the chronic demyelination. Interestingly, even if the mice are returned to a normal diet following 12 weeks of exposure to cuprizone, remyelination and oligodendrocyte regeneration does not occur. However, if adult O4+ progenitors are transplanted into the chronically demyelinated lesion of mice treated with cuprizone for 12 weeks, mature oligodendrocyte regeneration and remyelination occurs after the mice are returned to a normal diet. Thus, the formation of chronically demyelinated lesions induced by cuprizone appears to be the result of oligodendrocyte depletion within the lesion and not due to the inability of the chronically demyelinated axons to be remyelinated.

Niemann-Pick type C1 disease (NPC1) is an autosomal recessive neurovisceral storage disease caused by the mutation of NPC1 gene, resulting in perturbed intracellular transport of unesterified cholesterol. In NPC1, early-onset tauopathy is a constant feature. In addition, in NPC1 patients with ApoE epsilon4 homozygosity, deposition of A beta occurs mimicking Alzheimer disease (AD). Since AD is frequently associated with neuronal expression of alpha-synuclein, we investigated phosphorylated alpha-synuclein (psyn) immunoreactivity in the brains of 12 NPC1 patients, ages at death ranging from 9 months to 55 years. Psyn immunoreactivity was demonstrated in the perikarya of storage neurons and oligodendroglia in 10 cases. The immunoreactivity appeared more intense in subjects who had the ApoE epsilon4 allele. Lewy bodies were found in the substantia nigra in 2 of these cases. The psyn immunoreactivity was most intense in the substantia nigra where tauopathy was most severe. Phosphorylated tau and alpha-synuclein frequently colocalized. This study first documents alpha-synucleinopathy in NPC1. This observation suggests that the defect in intracellular cholesterol trafficking in NPC1 may provoke aberrant phosphorylation of alpha-synuclein and tau, and that this phosphorylation is enhanced by the ApoE epsilon4 allele. Thus, elucidation of metabolic pathways in NPC1 could provide clues to common mechanisms associated with neurodegeneration.

Haematogenous leucocytes enter the central nervous system (CNS) during diverse disorders of varied aetiologies. Understanding the trafficking cues that mediate CNS leucocyte infiltration might promote the development of flexible and selective means to modulate inflammation to achieve clinical benefit. The trafficking machinery of leucocytes has been elucidated during the past decade and consists of cell-surface adhesion molecules, chemoattractant cytokines (chemokines) and their receptors. Recent work in our laboratory characterized chemokine receptors found on T lymphocytes and monocytes in brain sections from subjects with one pathological subtype of multiple sclerosis (MS), an immune-mediated inflammatory demyelinating disease. In these tissues, the types 1 and 5 CC chemokine receptors (CCR1 and CCR5) were detected on perivascular monocytic cells whereas only CCR5 was present on parenchymal macrophages. The type 3 CXC chemokine receptor (CXCR3) was present on virtually all CD3-positive T cells. In the current study, we evaluated the expression of these receptors on the infiltrating cells present in cases of other inflammatory CNS disorders including those of dysimmune, infectious, neoplastic, and vascular aetiology. Perivascular and parenchymal monocytic cells expressed CCR1 in all cases and CXCR3 was consistently present on a substantial proportion of CD3+ T cells. The occurrence of CCR5 on parenchymal macrophages was much less uniform across the varied disorders. These data implicate CCR1 in monocyte infiltration of the CNS and are consistent with reports of studies in CCR1-deficient mice. CXCR3 is also likely to play a role in accumulation of T cells in the inflamed CNS. By contrast, our findings suggest that regulation of CCR5 on phagocytic macrophages may be contingent on the lesion environment.

Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, was shown previously to promote remyelination and oligodendrocyte precursor proliferation in a murine model for demyelination and remyelination. We used Affymetrix microarrays in this study to identify (1) changes in gene expression that accompany demyelination versus remyelination and (2) changes in gene expression during the successful remyelination of wild-type mice versus the unsuccessful attempts in mice lacking TNFalpha. Alterations in inflammatory genes represented the most prominent changes, with major histocompatibility complex (MHC) genes dramatically enhanced in microglia and astrocytes during demyelination, remyelination, and as a consequence of TNFalpha stimulation. Studies to examine the roles of these genes in remyelination were then performed using mice lacking specific genes identified by the microarray. Analysis of MHC-II-null mice showed delayed remyelination and regeneration of oligodendrocytes, whereas removal of MHC-I had little effect. These data point to the induction of MHC-II by TNFalpha as an important regulatory event in remyelination and emphasize the active inflammatory response in regeneration after pathology in the brain.

It is unknown why cerebellar neurons resist neurofibrillary tangle (NFT) formation. In Niemann-Pick disease Type C (NPC), NFT-mediated neurodegeneration occurs throughout brain, but the cerebellum degenerates conspicuously without NFT. To understand why, we have studied markers of NFT pathogenesis in cerebellum from 17 NPC cases, all having abundant NFT in forebrain. Remarkably, we found that NPC cerebella display several early markers of NFT formation, i.e., hyperphosphorylated tau and an array of cell cycle regulators, suggesting that cerebellar neurons in NPC undergo similar modifications as other neurons that develop NFT. However, cerebellar neurons are deficient in tau, the building block of NFT, and this may be one reason for their inability to form NFT. Even without NFT, cerebellar neurodegeneration may be triggered by the inappropriate activation of the cell cycle cdc2 kinase, and the npc-1 murine model provides an opportunity to test this hypothesis.

The contribution of peripheral macrophage was assessed in cuprizone intoxication, a model of demyelination and remyelination in which the blood-brain barrier remains intact. Flow cytometry of brain cells isolated from cuprizone-treated mice revealed an increase in the percentage of Mac-1(+)/CD45(hi) peripheral macrophage. To confirm these results in situ, C57BL/6 mice were lethally irradiated, transplanted with bone marrow from GFP-transgenic mice, and exposed to cuprizone. GFP(+) peripheral macrophages were seen in the CNS after 2 weeks of treatment, and infiltration continued through 6 weeks. While the peripheral macrophages were far outnumbered by the resident microglia, their recruitment across the blood-brain barrier alludes to a potentially important role.

Niemann-Pick type C disease is a neurovisceral storage disorder. Neurofibrillary tangles similar to those in Alzheimer's disease have been reported in most juvenile/adult patients without amyloid beta protein (Abeta) deposits. Recently, we found deposits of Abeta in the form of diffuse plaques in three (31- and 32-year-old sisters and a 37-year-old man) of nine Niemann-Pick type C disease patients, who presented with most severe tauopathy and with numerous neurofibrillary tangles. Abeta deposits were not detected in any of the control brains of patients younger than age 42 years. These three patients with Abeta deposit all were homozygotes of apolipoprotein E epsilon 4. Our study suggested that NPC1 gene mutations combined with homozygosity of apolipoprotein E epsilon 4 alleles could manifest neuropathology similar to that of Alzheimer's disease. Investigation of these patients may provide an important clue for understanding the pathogenesis of Alzheimer's disease.

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na(+) channels are clustered at high densities at the nodes, whereas shaker-type K(+) channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na(+) and K(+) channel clusters, altered nodal length, abnormal localization of K(+)channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na(+) channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na(+) channels.

The genetic demyelinating mouse "twitcher" is a model of the human globoid cell leukodystrophy, caused by galactosylceramidase (GALC) deficiency. Demyelination in the twitcher brain is secondary to apoptotic death of oligodendrocytes (OLs). Lipocalin-type prostaglandin (PG) D synthase (L-PGDS), a protein expressed in mature OLs, was progressively upregulated in twitcher OLs; whereas expression of OL-associated proteins such as carbonic anhydrase II, myelin basic protein, and myelin-associated glycoprotein was downregulated during demyelination in twitcher brains. The upregulation of L-PGDS was more remarkable in perineuronal OLs than in interfascicular OLs. A larger number of L-PGDS-positive OLs was found in selected fiber tracts of twitcher brains where fewer apoptotic cells were detected. The distribution of L-PGDS-positive OLs was inversely related to the severity of demyelination, as assessed by accumulation of scavenger macrophages. Mice doubly deficient for L-PGDS and GALC disclosed a large number of apoptotic neurons, which were never seen in twitcher brains, in addition to an increased number of apoptotic OLs. A linear positive correlation was observed between the population of L-PGDS-positive OLs in the twitcher brain and the ratio of apoptotic nuclei in the double mutant versus those in the twitcher, suggesting a dose-dependent effect of L-PGDS against apoptosis. These lines of evidence suggest that L-PGDS is an anti-apoptotic molecule protecting neurons and OLs from apoptosis in the twitcher mouse. This is a novel example of OL-neuronal interaction.

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.

Background Fabry disease is an X-linked lysosomal deficiency of α-galactosidase A that results in cellular accumulation of galacto-conjugates such as globotriosylceramide, particularly in blood vessels. It is associated with early-onset stroke and kidney and heart failure. Methods and Results Using [ 15 O] H 2 O and PET, we found increased resting regional cerebral blood flow in Fabry disease without evidence of occlusive vasculopathy or cerebral hypoperfusion. Because nitric oxide is known to play an important role in vascular tone and reactivity, we studied plasma nitrate, nitrite, and low-molecular-weight S -nitrosothiol levels by chemiluminescence. Skin biopsy specimens and archived brain tissue were also examined immunohistochemically for nitrotyrosine. Plasma nitrate, nitrite, and low-molecular-weight S -nitrosothiol were in the normal range; however, enhanced nitrotyrosine staining was observed in dermal and cerebral blood vessels. After a double-blind, placebo-controlled trial of α-galactosidase A therapy, the resting regional cerebral blood flow in the treated group was significantly reduced, with a notable decrease of nitrotyrosine staining in dermal blood vessels. Conclusions These findings suggest a chronic alteration of the nitric oxide pathway in Fabry disease, with critical protein nitration that is reversible with enzyme replacement therapy.

Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.

Exposure of young adult C57BL/6 mice to cuprizone in the diet initiated profound and synchronous demyelination of the corpus callosum, which was virtually complete by 4 weeks of exposure. Interestingly, even in the face of a continued exposure to cuprizone, there was spontaneous remyelination 2 weeks later. This remyelination preferentially involved smaller calibre axons. There was a suggestion of yet another cycle of demyelination (at 10 weeks) and remyelination (at 12 weeks), but by 16 weeks of exposure, the regenerative capacity was exhausted and the animals were near death. The relapsing-remitting pattern suggests this may be a useful model for certain human demyelinating disorders. In contrast to the above chronic model, the corpus callosum from mice exposed to cuprizone for only 6 weeks continued to remyelinate, with 67% of the axons being myelinated or remyelinated at 10 weeks. Interestingly, a significant reduction in the mean value for axonal diameter was observed during acute demyelination. Upon remyelination, however, the axonal calibre distribution returned to near-normal. In contrast, when mice were maintained on a cuprizone diet for 16 weeks, the mean value for axonal diameter was reduced to 60% of normal. These results provide further evidence that the interactions between oligodendrocytes and axons alter axonal calibre.

The axolemma is considered a well-established mitogen, responsible for Schwann cell proliferation during Wallerian degeneration in the peripheral nerve. However, very little is known about the role of macrophages in Schwann cell proliferation. To test the possible influence of macrophages on Schwann cell proliferation during Wallerian degeneration, macrophages were depleted by dichloromethylene diphosphonate (CI2MDP)-containing liposomes in two-month old C57BL/6J mice. CI2MDP-containing liposomes were injected into the mice intravenously prior to inducing Wallerian degeneration. The injection was repeated every other day to maintain macrophage depletion. Physiologic saline was injected into the control mice. To assess macrophage depletion in vitro, cells were isolated from sciatic nerves at 1, 2, 3, 5, and 7 days post-transection (DPT) and Mac-1 positive cells attached to coverslips were counted. In an in vivo study, Mac-1 positive cells were counted on sciatic nerve sections at the same time points. Throughout the course, the number of Mac-1 positive cells in macrophage-depleted mice was less than that in the control mice both in vivo and in vitro. Schwann cell proliferation was assessed by an in vitro system that reflects in vivo status at the time of cell isolation. Schwann cells were isolated from sciatic nerves at the same time points and proliferation rate was measured by thymidine autoradiography. The proliferation rate was mildly suppressed in macrophage-depleted mice, especially for the initial 3 DPT; however, the pattern of proliferation was not significantly different from controls. These results suggest that macrophages contribute to Schwann cell proliferation during Wallerian degeneration however, their contribution may be relatively limited.

The twitcher mouse is a murine model of globoid cell leukodystropy, a genetic demyelinating disease caused by a mutation of the galactosylceramidase gene. Demyelination of the central nervous system commences around 20 postnatal days. Using GFP-transgenic mice as donors, the distribution of hematogenous cells after bone marrow transplantation was investigated in the twitcher mice. Bone marrow transplantation was carried out at 8 postnatal days. In twitcher chimeric mice examined before 30 postnatal days, numerous GFP(+) cells were detected in spleen and peripheral nerve but only a few were detected in the liver, lung, and spinal white matter. In contrast, at 35 to 40 postnatal days when demyelination is evident, many GFP(+) cells with ameboid form were detected in the white matter of the spinal cord, brainstem, and cerebrum. Approximately half of these GFP(+) cells were co-labeled with Mac-1. In twitcher chimeric mice examined after 100 postnatal days, the majority of GFP/Mac-1 double-positive cells displayed the morphological features of ramified microglia with fine delicate processes and was distributed diffusely in both gray and white matter. These results suggest that a significant number of donor hematogenous cells are able to infiltrate into the brain parenchyma, repositioning themselves into areas previously occupied by microglia, and to ameliorate lethality.

Niemann-Pick disease Type C (NP-C) is a progressive neurodegenerative disorder caused by mutations in the NPC1 gene and characterized by intracellular accumulation of cholesterol and sphingo-lipids. The major neuronal storage material in NP-C consists of gangliosides and other glycolipids, raising the possibility that the accumulation of these lipids may participate in the neurodegenerative process. To determine if ganglioside accumulation is a crucial factor in neuropathogenesis, we bred NP-C model mice with mice carrying a targeted mutation in GalNAcT, the gene encoding the beta-1-4GalNAc transferase responsible for the synthesis of GM2 and complex gangliosides. Unlike the NP-C model mice, these double mutant mice did not exhibit central nervous system (CNS) accumulation of gangliosides GM2 or of glycolipids GA1 and GA2. Histological analysis revealed that the characteristic neuronal storage pathology of NP-C disease was substantially reduced in the double mutant mice. By contrast, visceral pathology was similar in the NP-C and double mutant mice. Most notably, the clinical phenotype of the double mutant mice, in the absence of CNS ganglioside accumulation and associated neuronal pathology, did not improve. The results demonstrate that complex ganglioside storage, while responsible for much of the neuronal pathology, does not significantly influence the clinical phenotype of the NP-C model.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc-/-, cgt+/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt+/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7 ± 3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9 ± 1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc-/-, cgt+/- mice from galc-/-, cgt+/+ mice. The brain psychosine level in galc-/-, cgt+/- mice was also approximately two-thirds of the galc-/-, cgt+/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies. J. Neurosci. Res. 59:179–187, 2000 © 2000 Wiley-Liss, Inc.

Tissue distribution of beta-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl beta-D-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (Hexa-/-, Hexb-/-, Gm2a-/- and Hexa-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of beta-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and Hexa-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or Hexa-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/- +WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.

We have documented changes in the oligodendrocyte population during demyelinating insult to the adult CNS. Feeding of cuprizone to adult mice led to apoptotic death of mature oligodendrocytes followed by profound demyelination of the corpus callosum. A regenerative response was initiated even during active demyelination. Oligodendrocyte progenitors have begun to proliferate and then accumulate within the lesion. Many of these cells may have migrated from the sub-ventricular zone and fornix before their accumulation in the demyelinating corpus callosum. The accumulation of differentiating oligodendrocyte progenitors was followed closely by the reappearance of mature oligodendrocytes and remyelination. Interestingly, an increase in IGF-1 mRNA was detected at Week 3 through Week 7, suggesting potential involvement in remyelination. Other factors, however, such as PDGF, NT3, FGF, jagged, and notch remained unchanged. These results suggest that the mature oligodendroglial population depleted by apoptosis is replaced by a newly formed oligodendroglial population derived from progenitors; these accumulate and seem to differentiate during remyelination.

Morphological alterations of oligodendrocytes (OLs) leading to their depletion were studied in the genetic demyelinating mutant, twitcher, a murine model of globoid cell leukodystrophy (GLD). With pi-glutathione-S-transferase immunostaining, OLs with multiple varicose processes were recognized in the early stages and adjacent areas of demyelination and then the OLs cytoplasm as well as the processes became shrunken with progression of the disease. These shrunken OLs were labeled by the TUNEL method, indicative of apoptotic cell death. The ultrastructural features of apoptotic cells were noted in these OLs and DNA laddering was noted in the twitcher brain in advanced stages. This is the first report describing the gradual depletion of OLs by apoptosis in genetic demyelination.

Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff ’s disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.

To explore the feasibility of cell type-specific gene expression in oligodendrocytes as a possible therapeutic approach for demyelinating diseases, the cell specificity, tissue specificity, and duration of gene expression were investigated using recombinant adeno-associated viral vectors (rAAV) carrying a green fluorescence protein (GFP) gene. Recombinant AAV vectors carrying either the myelin basic protein (MBP) promoter (rAAV-MBP-GFP) or the cytomegalovirus (CMV) immediate early promoter (rAAV-CMV-GFP) were semistereotactically injected into the brain of C57BL/6J mice. Injection of the rAAV-MBP-GFP vector into or near the corpus callosum resulted in high levels of GFP expression in white matter regions. Double immunostaining with cell- specific markers proved that these GFP-expressing cells were oligodendrocytes. Injection of the rAAV- MBP-GFP vector into gray matter rarely produced GFP expression. In contrast, injection of the rAAV-CMV-GFP vector resulted in few GFP-expressing cells in the white matter, with most of the GFP-expressing cells being neurons located in the cerebral cortex along the needle track. The expression of the GFP driven by the MBP promoter persisted for at least 3 months.

Protective protein/cathepsin A (PPCA) is a pleiotropic lysosomal enzyme that complexes with beta-galactosidase and neuraminidase, and possesses serine carboxypeptidase activity. Its deficiency in man results in the neurodegenerative lysosomal storage disorder galactosialidosis (GS). The mouse model of this disease resembles the human early onset phenotype and results in severe nephropathy and ataxia. To understand better the pathophysiology of the disease, we compared the occurrence of lysosomal PPCA mRNA and protein in normal adult mouse tissues with the incidence of lysosomal storage in PPCA(-/-) mice. PPCA expression was markedly variable among different tissues. Most sites that produced both mRNA and protein at high levels in normal mice showed extensive and overt storage in the knockout mice. However, this correlation was not consistent as some cells that normally expressed high levels of PPCA were unaffected in their storage capability in the PPCA(-/-) mice. In addition, some normally low expressing cells accumulated large amounts of undegraded products in the GS mouse. This apparent discrepancy may reflect a requirement for the catalytic rather than the protective function of PPCA and/or the presence of cell-specific substrates in certain cell types. A detailed map showing the cellular distribution of PPCA in nomal mouse tissues as well as the sites of lysosomal storage in deficient mice is critical for accurate assessment of the effects of therapeutic interventions.

Effects of macrophage suppression on Schwann cell proliferation in Wallerian degeneration was investigated in vitro with thymidine autoradiography. Sciatic nerves of C57BL mice were transected shortly after an intraperitoneal injection of silica dust and Schwann cells were harvested at 1, 2, 3, 5, 7, and 10 days post-transection. In silica treated mice, Schwann cells from post-transected nerve stumps proliferated despite marked suppression of macrophages, although there was some initial delay. The results of this study suggest that the mechanism of Schwann cell proliferation is multifactorial and in addition to the influence of growth factors mediated and released by macrophages, other factor(s) such as degenerated axolemma may play a more pivotal role in Schwann cell proliferation in vivo.

Only a limited number of genetic mouse models of abnormal sphingolipid metabolism are known to occur spontaneously. However, recent progress in the combined homologous recombination and embryonic stem cell technology allows inactivation of any genes of choice once they are cloned. Not only is it possible to generate mutant mouse lines that are equivalent to known human genetic disorders but genetic conditions unknown or highly unlikely to occur in humans, such as simultaneous inactivation of more than one gene, can also be created. Most of the human disorders due to genetic defects in sphingolipid catabolism have been duplicated in the mouse. With increasing activity in cloning of the enzymes involved in sphingolipid biosynthesis, genetic mouse models of abnormal sphingolipid biosynthesis are beginning to appear. These models have already provided invaluable insight into the metabolism and physiological functions of sphingolipids and are expected to be utilized extensively for evaluation of the pathogenesis and of treatment approaches of these genetic disorders.

The GM2 gangliosidoses are a group of severe, neurodegenerative conditions that include Tay-Sachs disease, Sandhoff disease, and the GM2 activator deficiency. Bone marrow transplantation (BMT) was examined as a potential treatment for these disorders using a Sandhoff disease mouse model. BMT extended the life span of these mice from approximately 4.5 mo to up to 8 mo and slowed their neurologic deterioration. BMT also corrected biochemical deficiencies in somatic tissues as indicated by decreased excretion of urinary oligosaccharides, and lower glycolipid storage and increased levels of beta-hexosaminidase activity in visceral organs. Even with neurologic improvement, neither clear reduction of brain glycolipid storage nor improvement in neuronal pathology could be detected, suggesting a complex pathogenic mechanism. Histological analysis revealed beta-hexosaminidase-positive cells in the central nervous system and visceral organs with a concomitant reduction of colloidal iron-positive macrophages. These results may be important for the design of treatment approaches for the GM2 gangliosidoses.

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.

The G M2 activator deficiency (also known as the AB variant), Tay–Sachs disease, and Sandhoff disease are the major forms of the G M2 gangliosidoses, disorders caused by defective degradation of G M2 ganglioside. Tay–Sachs and Sandhoff diseases are caused by mutations in the genes ( HEXA and HEXB ) encoding the subunits of β-hexosaminidase A. The G M2 activator deficiency is caused by mutations in the GM2A gene encoding the G M2 activator protein. For degradation of G M2 ganglioside by β-hexosamindase A, the G M2 activator protein must participate by forming a soluble complex with the ganglioside. In each of the disorders, G M2 ganglioside and related lipids accumulate to pathologic levels in neuronal lysosomes, resulting in clinically similar disorders with an onset in the first year of life, progressive neurodegeneration, and death by early childhood. We previously have described mouse models of Tay–Sachs ( Hexa −/−) and Sandhoff ( Hexb −/−) diseases with vastly different clinical phenotypes. The Hexa −/− mice were asymptomatic whereas the Hexb −/− mice were severely affected. Through gene disruption in embryonic stem cells we now have established a mouse model of the G M2 activator deficiency that manifests an intermediate phenotype. The Gm2a −/− mice demonstrated neuronal storage but only in restricted regions of the brain (piriform, entorhinal cortex, amygdala, and hypothalamic nuclei) reminiscent of the asymptomatic Tay–Sachs model mice. However, unlike the Tay–Sachs mice, the Gm2a −/− mice displayed significant storage in the cerebellum and defects in balance and coordination. The abnormal ganglioside storage in the Gm2a −/− mice consisted of G M2 with a low amount of G A2 . The results demonstrate that the activator protein is required for G M2 degradation and also may indicate a role for the G M2 activator in G A2 degradation.

The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (alpha-subunit) and HEXB (beta-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, beta-hexosaminidase A (alpha beta), as well as for the homodimers beta-hexosaminidase B (beta beta) and S (alpha alpha). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs (Hexa-/-) and Sandhoff (Hexb-/-) disease model mice. Lacking both the alpha and beta-subunits these 'double knockout' mice displayed a total deficiency of all forms of lysosomal beta-hexosaminidase including the small amount of beta-hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients. This result demonstrates that glycosaminoglycans are crucial substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase enzyme system.

The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane greatly enriched in the galactolipid galactocerebroside (GalC) and its sulfated derivative sulfatide. We have generated mice lacking the enzyme UDP-galactose:ceramide galactosyltransferase (CGT), which is required for GalC synthesis. CGT-deficient mice do not synthesize GalC or sulfatide but surprisingly form myelin containing glucocerebroside, a lipid not previously identified in myelin. Microscopic and morphometric analyses revealed myelin of normal ultrastructural appearance, except for slightly thinner sheaths in the ventral region of the spinal cord. Nevertheless, these mice exhibit severe generalized tremoring and mild ataxia, and electrophysiological analysis showed conduction deficits consistent with reduced insulative capacity of the myelin sheath. Moreover, with age, CGT-deficient mice develop progressive hindlimb paralysis and extensive vacuolation of the ventral region of the spinal cord. These results indicate that GalC and sulfatide play important roles in myelin function and stability.