Parkinsonism among Gaucher disease carriers.

An association between Gaucher disease and Parkinson disease has been demonstrated by the concurrence of Gaucher disease and parkinsonism in rare patients and the identification of glucocerebrosidase mutations in probands with sporadic Parkinson disease. Using a different and complementary approach, we describe 10 unrelated families of subjects with Gaucher disease where obligate or confirmed carriers of glucocerebrosidase mutations developed parkinsonism. These observations indicate that mutant glucocerebrosidase, even in heterozygotes, may be a risk factor for the development of parkinsonism. Understanding the relationship between altered glucocerebrosidase and the development of parkinsonian manifestations will provide insights into the genetics, pathogenesis, and treatment of Parkinson disease.

Gaucher disease with parkinsonian manifestations: does glucocerebrosidase deficiency contribute to a vulnerability to parkinsonism?

Among the phenotypes associated with Gaucher disease, the deficiency of glucocerebrosidase, are rare patients with early onset, treatment-refractory parkinsonism. Sequencing of glucocerebrosidase in 17 such patients revealed 12 different genotypes. Fourteen patients had the common "non-neuronopathic" N370S mutation, including five N370S homozygotes. While brain glucosylsphingosine levels were not elevated, Lewy bodies were seen in the four brains available for study. The shared clinical and neuropathologic findings in this subgroup suggest that the deficiency in glucocerebrosidase may contribute to a vulnerability to parkinsonism.

The E326K mutation and Gaucher disease: mutation or polymorphism?

Gaucher disease is caused by mutations in the gene for human glucocerebrosidase, a lysosomal enzyme involved in the intracellular hydrolysis of glucosylceramide. While over 150 different glucocerebrosidase mutations have been identified in patients with Gaucher disease, not all reported mutations have been fully characterized as being causative. One such mutation is the E326K mutation, which results from a G to A nucleotide substitution at genomic position 6195 and has been identified in patients with type 1, type 2 and type 3 Gaucher disease. However, in each instance, the E326K mutation was found on the same allele with another glucocerebrosidase mutation. Utilizing polymerase chain reaction (PCR) screening and restriction digestions of both patients with Gaucher disease and normal controls, we identified the E326K allele in both groups. Of the 310 alleles screened from patients with Gaucher disease, the E326K mutation was detected in four alleles (1.3%). In addition, screening for the E326K mutation among normal controls from a random population revealed that three alleles among 316 screened (0.9%) also carried the E326K mutation. In the normal controls with the E326K allele, the glucocerebrosidase gene was completely sequenced, but no additional mutations were found. Because the E326K mutation may be a polymorphism, we caution that a careful examination of any allele with this mutation should be performed to check for the presence of other glucocerebrosidase mutations.

Glucosylsphingosine accumulation in mice and patients with type 2 Gaucher disease begins early in gestation.

Gaucher disease, the most common of the sphingolipidoses, results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although type 2 (acute neuronopathic) Gaucher disease is associated with rapidly progressive and fatal neurologic deterioration, the pathophysiologic mechanisms leading to the neurologic symptoms and early demise remain uncharacterized. While the pathology encountered in Gaucher disease has been attributed to glucocerebroside storage, glucosylsphingosine (Glc-sph), a cytotoxic compound, also accumulates in the tissues. Elevations of brain Glc-sph have been reported in patients with types 2 and 3 Gaucher disease. In this study, Glc-sph levels were measured using HPLC in tissues from mice with type 2 Gaucher disease created with a null glucocerebrosidase allele. Compared with unaffected littermates, homozygous mice with type 2 Gaucher disease had approximately a 100-fold elevation of Glc-sph in brain, as well as elevated levels in other tissues. This accumulation was detected in utero by E 13 and increased progressively throughout gestation. Similarly, elevated Glc-sph levels were seen in human fetuses with type 2 Gaucher disease, indicating that therapy initiated after birth may be too late to prevent the sequelae of progressive neurologic damage that begins early in gestation. These findings suggest that the accumulation of Glc-sph may be responsible for the rapid demise of mice with type 2 Gaucher disease and the devastating clinical course seen in patients with type 2 Gaucher disease.

Monitoring the CNS pathology in aspartylglucosaminuria mice.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal storage disorder caused by the deficiency of the aspartylglucosaminidase (AGA) enzyme. The hallmark of AGU is slowly progressing mental retardation but the progression of brain pathology has remained uncharacterized in humans. Here we describe the long-term follow-up of mice carrying a targeted AGU-mutation in both alleles. Immunohistochemistry, histology, electron microscopy, quantitative magnetic resonance imaging (MRI) and behavioral studies were carried out to evaluate the CNS affection of the disease during development. The lysosomal storage vacuoles of the AGA -/- mice were most evident in central brain regions where MRI also revealed signs of brain atrophy similar to that seen in the older human patients. By immunohistochemistry and MRI examinations, a subtle delay of myelination was observed in AGA -/- mice. The life span of the AGA -/- mice was not shortened. Similar to the slow clinical course observed in human patients, the AGA -/- mice have behavioral symptoms that emerge at older age. Thus, the AGU knock-out mice represent an accurate model for AGU, both histopathologically and phenotypically.

Mice with an aspartylglucosaminuria mutation similar to humans replicate the pathophysiology in patients.

Aspartyglucosaminuria (AGU) is a lysosomal storage disease with autosomal recessive inheritance that is caused by deficient activity of aspartylglucosaminidase (AGA), a lysosomal enzyme belonging to the newly described enzyme family of N-terminal hydrolases. An AGU mouse model was generated by targeted disruption of the AGA gene designed to mimic closely one human disease mutation. These homozygous mutant mice have no detectable AGA activity and excrete aspartylglucosamine in their urine. Analogously to the human disease, the affected homozygous animals showed storage in lysosomes in all analyzed tissues, including the brain, liver, kidney and skin, and lysosomal storage was already detected in fetuses at 19 days gestation. Electron microscopic studies of brain tissue samples demonstrated lysosomal storage vacuoles in the neurons and glia of the neocortical and cortical regions. Magnetic resonance images (MRI) facilitating monitoring of the brains of living animals indicated cerebral atrophy and hypointensity of the deep gray matter structures of brain-findings similar to those observed in human patients. AGU mice are fertile, and up to 11 months of age their movement and behavior do not differ from their age-matched littermates. However, in the Morris water maze test, a slow worsening of performance could be seen with age. The phenotype mimics well AGU in humans, the patients characteristically showing only slowly progressive mental retardation and relatively mild skeletal abnormalities.

Targeted reduction of oxytocin expression provides insights into its physiological roles.

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 hours without milk in their stomachs. OT injection into the dams or rescue with the rat OT gene restores the milk ejection in response to suckling. OT is also needed for post-partum alveolar proliferation. These results indicate an absolute requirement for oxytocin for successful milk ejection, but not for mating, parturition and milk production, in mice. Furthermore, homozygous mutant mice show reduced aggression in some tests.

Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition.

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 h without milk in their stomachs. OT injection into the dams restores the milk injection in response to suckling. These results indicate an absolute requirement for oxytocin for successful milk injection, but not for mating, parturition and milk production, in mice.

Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.

We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease.

A biochemical and ultrastructural evaluation of the type 2 Gaucher mouse.

Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficient in glucocerebrosidase and have a rapidly deteriorating clinical course analogous to the most severely affected type 2 human patients. An ultrastructural study of tissues from these mice revealed glucocerebroside accumulation in bone marrow, liver, spleen, and brain. This glycolipid had a characteristic elongated tubular structure and was contained in lysosomes, as demonstrated by colocalization with both ingested carbon particles and cathepsin D. In the central nervous system (CNS), glucocerebroside was diffusely stored in microglia cells and in brainstem and spinal cord neurons, but not in neurons of the cerebellum or cerebral cortex. This rostralcaudal pattern of neuronal lipid storage in these Gaucher mice replicates the pattern seen in type 2 human Gaucher patients and clearly demonstrates that glycosphingolipid catabolism and/or accumulation varies within different brain regions. Surprisingly, the cellular pathology of tissue from these Gaucher mice was relatively mild, and suggests that the early and rapid demise of both Gaucher mice and severely affected type 2 human neonates may be the result of both a neurotoxic metabolite, such as glucosylsphingosine, and other factors, such as skin water barrier dysfunction secondary to the absence of glucocerebrosidase activity.

Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene.

Gaucher's disease is the most prevalent lysosomal storage disorder in humans and results from an autosomally inherited deficiency of the enzyme glucocerebrosidase (beta-D-glucosyl-N-acylsphingosine glucohydrolase), which is responsible for degrading the sphingolipid glucocerebroside. An animal model for Gaucher's disease would be important for investigating its phenotypic diversity and pathogenesis and for evaluating therapeutic approaches. A naturally occurring canine model has been reported but not propagated. Attempts to mimic the disease in animals by inhibiting glucocerebrosidase have been inadequate. Here we generate an animal model for Gaucher's disease by creating a null allele in embryonic stem cells through gene targeting and using these genetically modified cells to establish a mouse strain carrying the mutation. Mice homozygous for this mutation have less than 4% of normal glucocerebrosidase activity, die within twenty-four hours of birth and store glucocerebroside in lysosomes of cells of the reticuloendothelial system.

Brain grafts and Parkinson's disease.

In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.

Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector.

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.

Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in the type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is approximately equal to 80% informative in all Gaucher patients studied.

Glycosylation and processing of high levels of active human glucocerebrosidase in invertebrate cells using a baculovirus expression vector.

A human cDNA containing the complete coding region for the lysosomal glycoprotein glucocerebrosidase (EC 3.2.1.45) was introduced into the genome of Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus produced high levels of glucocerebrosidase, 40% of which was in the culture medium. The amino-terminal amino acid sequence of the recombinantly produced enzyme was identical to that of mature, human placental glucocerebrosidase, demonstrating that the signal sequence of the human preenzyme was recognized and appropriately removed in the SF9 invertebrate cells. The glucocerebrosidase in both the culture supernatant and SF9 cell pellet was glycosylated and contained, in part, high mannose oligosaccharide. These results demonstrate that insect cells can be used to produce abundant quantities of active mature human glucocerebrosidase that contains high mannose oligosaccharide as a consequence of post-translational processing.

Retrovirus-mediated transfer of the human glucocerebrosidase gene to Gaucher fibroblasts.

A recombinant retrovirus carrying complementary DNA containing the complete coding sequence for human glucocerebrosidase was constructed and used to infect mouse cells and human fibroblasts from a type 2 Gaucher patient. The cDNA stably integrated in the genome of the recipient cell. Infected cells made glucocerebrosidase-cross-reacting material, and the protein was glycosylated and contained the epitope(s) missing in the type 2 mutant glucocerebrosidase. This study demonstrates the feasibility of efficiently transferring the gene encoding glucocerebrosidase to Gaucher cells using retrovirus as vector.

Demonstration of remarkable sequence divergence in variants of a complex satellite DNA by molecular cloning.

Repeat units of a complex G + C-rich satellite of the Bermuda land crab have been cloned by insertion into either the PstI or EcoRI site of pBR322 or the EcoRI site of pUC9. While most of the recombinants contained inserts of approx. 2.1 kb, the average size of repeat units seen in cellular satellite digests, several inserts were markedly different in size. Two domains that account for major sequence differences among the satellite variants and that may be 'hotspots' for sequence modification have been subcloned to permit characterization of their secondary and tertiary structures independent of the influence of the other unusual sequences present. One of these domains is striking in its content of simple repeats; one strand is highly biased in pyrimidines which may permit the formation of unusual secondary and/or tertiary conformations. The other subcloned domain is rich in Pu/Py; preliminary data indicate a transition from B----Z DNA in this region.

Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA.

The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.