Gaucher's disease: a paradigm for interventional genetics

Gaucher's disease (GD) is one of the most prevalent lysosomal storage disorders (LSDs) and a rare genetic disease for which specific therapy is now available. GD is an autosomal, recessive, inborn error of glycosphingolipid metabolism, due to a deficiency in the enzyme acid β-glucosidase. Partial deficiency of acid β-glucosidase is associated with parenchymal disease of the liver, spleen, and bone marrow with concomittant anemia and thrombocytopenia in non-neuronopathic, type 1 GD. Severe deficiency of glucocerebrosidase caused by severe mutations is additionally associated with neurological manifestations in the less common type 2 and type 3 GD subtypes. Outside of the Ashkenazi Jewish community, a high molecular diversity is observed. Clarification of genotype/phenotype relationship and the identification of modifier loci that impact on GD phenotypes remains a critical area for research. Enzyme replacement therapy (ERT) is proven to be safe and effective in the treatment of type 1 GD, establishing imiglucerase as the current standard of care. Amelioration of hepatosplenomegaly and of hematological manifestations is usually apparent within 6–12 months, whereas the bone disease responds more slowly. ERT cannot reverse the neurological deficits in type 2 or type 3 GD. Small molecule inhibitors of glucosylceramide synthase are being developed for substrate reduction therapy. Other potential therapeutic options such as chaperon-mediated enzyme enhancement therapy and gene therapy are being explored.     

Pathogenesis of brain dysfunction in Batten disease

Animal models of Batten disease and other neuronal storage disorders offer important opportunities to study the pathogenesis of brain dysfunction in this family of diseases. Although all of these conditions exhibit progressive intraneuronal storage, we have found that other aspects of the cellular pathology of Batten disease differ markedly from those of storage disorders caused by lysosomal hydrolase deficiencies. Likewise, atrophy of cerebral cortex and other select brain regions, a prominent characteristic of Batten disease, does not occur in most other storage disorders. Our studies indicate that Batten disease has findings in common with human neurodegenerative diseases and that neuron death may be caused by excitotoxicity occurring secondary to the combined effects of suboptimal mitochondrial function and GABAergic (inhibitory) cell loss. © 1995 Wiley-Liss, Inc.     

Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage with a wide spectrum of clinical phenotypes. At the cellular level, the disorder is characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system. Approximatively 95% of patients have mutations in the NPC1 gene (mapped at 18q11) which encodes a large membrane glycoprotein primarily located to late endosomes. The remainder have mutations in the NPC2 gene (mapped at 14q24.3) which encodes a small soluble lysosomal protein with cholesterol-binding properties. The identical biochemical patterns observed in NPC1 and NPC2 mutants suggest that the two proteins function in a coordinate fashion. Identification of mutations revealed a complex picture of molecular heterogeneity, allowing genotype - phenotype correlations for both genes and providing insights into structure - function relationships for the NPC1 protein. Although a whole body of evidence suggests that the NPC1 and NPC2 proteins are involved in the cellular postlysosomal/late endosomal transport of cholesterol, glycolipids and other cargo, their precise functions and relationship remain unclear and are currently the subject of intense investigation. These studies, conducted in various models, should ultimately lead to a better understanding of the pathophysiology of NPC and new therapeutic approaches.     

Stored dolichyl pyrophosphoryl oligosaccharides in Batten disease

Each of the 3 childhood forms of Batten disease, juvenile (JB), late-infantile (LIB), and infantile (IB), have abnormally high brain concentrations of dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). In this study, the carbohydrate portions of Dol-PP-OS were analysed: in JB and LIB, they range in size from Man₂GlcNAc₂ to Glc₃Man₉GlcNAc₂, predominant components being Man_5–7GlcNAc₂ and Glc₃Man₇GlcNAc₂. In IB, they range from Man_6–9GlcNAc₂, no glucose containing oli-gosacchsrides being identified. In Batten disease, the main subcellular location of Dol-PP-OS is within storage material, where it represents up to 7% of the dry weight. [³H]-Mannose incorporation experiments with cultured fibroblasts show that synthesis of Dol-PP-OS in JB is normal. We infer that the glycosyla-tion intermediate Glc₃Man₉GlcNAc₂-PP-dol-ichol is synthesised normally within the endo-plasmic reticulum in Batten disease, but that catabolic derivatives accumulate within the lysosomes. It is unclear whether this process is central to the pathogenesis of the disease, though in IB a defect in the release of man-nose residues from Dol-PP-OS is a distinct possibility.     

Changes of melanosome morphology associated with the differentiation of epidermal melanocytes in slaty mice

The slaty (Dct^slt) mutation is known to reduce the activity of dopachrome tautomerase, which converts dopachrome to 5,6‐dihydroxyindole‐2‐carboxylic acid in the pathway of eumelanin synthesis and to inhibit melanosome maturation in melanocytes. However, it is not known whether the inhibition of melanosome maturation in slaty melanocytes is developmentally regulated. To address this point, changes in the morphology and maturation of melanosomes in cultured epidermal melanocytes derived from newborn mice of wild‐type (black) and slaty mutant were surveyed under the electron microscope. In black melanocytes (Dct⁺), almost all melanosomes were elliptical stage IV melanosomes. However, in slaty melanocytes, numerous spherical stage III melanosomes with globular depositions of pigment in addition to elliptical stage III melanosomes with intraluminal fibrils were observed. Mixed‐type melanosomes containing both globular deposition and intraluminal fibrils of pigment were also observed. In slaty melanocytes, spherical and mixed‐type melanosomes were gradually decreased after birth, whereas elliptical melanosomes were gradually increased. Stage IV melanosomes were very few in slaty melanocytes, and the number did not increase after birth. These results suggest that the slaty mutation blocks the melanosome maturation at stage III and affects the melanosome morphology (elliptical or spherical) in a developmental stage‐specific manner. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

原代培养小鼠皮层神经细胞对淀粉样蛋白的内化作用

目的：观察原代培养神经细胞对淀粉样蛋白（β-amyloid protein,Aβ）内化,以及星形胶质细胞对这一过程的影响。方法：纯小鼠皮质神经细胞培养14d,分成对照组和Aβ组,各组再分为3个亚组,分别用3种不同浓度的荧光素或荧光素标记的淀粉样蛋白1-42（Aβ1-42-fluo）与神经细胞共孵育24h,在激光扫描共聚焦显微镜下直接观察或进行免疫荧光多重染色后镜下观察,结合图像分析方法分析神经细胞内化Aβ情况及亚细胞结构定位;另取小鼠皮质神经细胞与星形胶质细胞共培养14d,分成对照组和Aβ组,各组分别与200nmol/L荧光素或200nmol/L Aβ1-42-fluo共孵育24h,采用上述方法分析比较星形胶质细胞对神经细胞内化Aβ的影响。结果：荧光素各组细胞内均未见荧光颗粒。培养的纯神经细胞24h内能将浓度为100和200nmol/L Aβ1-42-fluo内化入细胞内,荧光颗粒在神经细胞的胞体和突起均有分布。内化作用与Aβ浓度相关,经免疫荧光方法证明部分内化Aβ位于溶酶体内;在神经细胞与星形胶质细胞共培养组中,神经细胞内化Aβ较纯神经细胞培养组明显增加（P〈0.05）。结论：神经细胞能内化一定浓度的Aβ,星形胶质细胞具有促进神经细胞内化Aβ的作用。     

Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders

The mucopolysaccharidoses (MPSs) are a complex family of lysosomal storage disorders characterized by failure to degrade heparan sulfate (HS) and/or other types of glycosaminoglycans (GAGs) secondary to the absence of specific lysosomal enzymes. An accompanying storage of glycosphingolipids (GSLs), most notably GM2 and GM3 gangliosides, has also been documented to occur in many types of MPS disease and is believed to be caused by secondary inhibition of GSL-degradative enzymes by intracellular GAG accumulation. We have documented the presence of secondary ganglioside accumulation in mouse models of several MPS disorders (types I, IIIA, IIIB, and VII) and report that this storage is accompanied by sequestration of free cholesterol in a manner similar to that observed in primary gangliosidoses. Using confocal microscopy, we evaluated the cellular distribution of cholesterol, GM2 and GM3 gangliosides, and HS in brains of mice with MPS IIIA disease. Unexpectedly, we found that although both gangliosides often accumulated in the same neurons, they were consistently located in separate populations of cytoplasmic vesicles. Additionally, GM3 ganglioside only partially co-localized with the primary storage material (HS), and cholesterol likewise only partially co-localized with the GM2 and GM3 gangliosides. These findings raise significant questions about the mechanism(s) responsible for secondary accumulation of storage materials in MPS disease. Furthermore, given that GSLs and cholesterol are constituents of membrane rafts believed critical in signal transduction events in neurons, their co-sequestration in individual neurons suggests the presence of defects in the composition, trafficking, and/or recycling of raft components and thus possible new mechanisms to explain neuronal dysfunction in MPS disorders.     

Spatial patterns of mammalian brain aging: distribution of cathepsin D-immunoreactive cell bodies and dystrophic dendrites in aging dogs resembles that in Alzheimer's disease

Elevated levels of the lysosomal enzyme cathepsin D are found in the early stages of Alzheimer's disease (AD) and co-occur with intraneuronal tangles. The present study tested whether increases in cathepsin D would emerge during aging in another mammalian species. Regional brain patterns of cathepsin D immunostaining were compared in dogs ages 0.35 to 16 years. Accumulations of immunopositive material were evident in neuronal cell bodies in many forebrain sites in middle-age to old dogs (>/=6 years). Three types could be distinguished: (1) dense aggregates with no particular position within the cell body; (2) crescent-shaped "caps" that occupied one pole of the cell body; and (3) very dense "spikes" that extended from the cell body for variable distances into the apical dendrite; these spikes were found in only a few areas, most notably the subiculum and layer V of neocortex. The spikes appeared between ages 2 and 5 years and increased steadily with age thereafter. Spikes were found in the subiculum in the aged human brain but only infrequently; they were, however, present in large numbers in AD brains. These results established that brain aging in dogs is (1) well advanced by middle age, (2) varies markedly across regions, and (3) in at least some of its aspects (dystrophic dendrites) is prominent in areas known to exhibit pathology early in the course of AD. Combined with previous results for rats, these findings indicated that changes in cathepsin D observed in AD, in particular in the temporal lobe, reflect a generalized mammalian pattern of brain aging.     

Temporal and spatial profiles of ABCA2-expressing oligodendrocytes in the developing rat brain

ABCA2 protein belongs to the ABCA subclass of ATP-binding cassette (ABC) transporters proposed to exert critical functions in transmembrane transport of endogenous lipids. In this study, we found by immunoblot analyses that approximately 260 kDa of ABCA2 protein is expressed predominantly in oligodendrocytes, and that the expression of the protein is upregulated in the brain during maturation, especially between postnatal days 6 and 19. Parallel to the changes in expression of ABCA2, immunohistochemical analyses showed rapid spatial spread of ABCA2-immunolabeled oligodendrocytes in the brain during this period. These temporal and spatial changes in ABCA2 expression were in good agreement with findings in myeloarchitectonics reported previously. Further, double immunolabeling with ABCA2 and a major structural protein of myelin, myelin basic protein, demonstrated that onset of ABCA2 expression in oligodendrocytes coincides with the appearance of thick myelin segments immunolabeled with myelin basic protein. Because ABCA2 was abundantly expressed in adult cortex in white matter and gray matter, coexpression of ABCA2 and a marker for the oligodendroglial progenitors NG2 or platelet-derived growth factor alpha receptor was investigated. No cells coexpressing ABCA2 and the marker were observed, suggesting that ABCA2 is expressed predominantly in myelin-forming oligodendrocytes distinct from the adult oligodendroglial progenitors tested. These results suggested a role for ABCA2 in membrane transport of substrates such as the lipids that are closely linked to myelination processes.     

Effects of lysosomotropic agents,cytochalasin B and colchicine on the “down-regulation” of prolactin receptors in mammary gland explants

Effects of lysosomotropic agents and drugs which disrupt the cytoskeleton on prolactin receptor levels were studied in organ culture of rabbit mammary glands. As we have shown previously, prolactin is able to induce a down-regulation of its own receptor levels. In the present experiments, lysosomotropic agents, chloroquine, ammonium chloride and methylamine, in the presence of prolactin are capable of almost completely preventing this down-regulation. Interestingly, these lysosomotropic agents alone could increase the level of prolactin receptors, which confirms that the degradation of prolactin receptors occurs in the lysosomes and that this is a rapid process which can proceed in the absence of prolactin. The almost complete lack of effect of microfilament (cytochalasin B) or microtubule (colchicine) disrupting drugs seems to indicate that the cytoskeleton is not involved in the down-regulation process.