Inborn Lysosomal Disease Research Articles

Zebrafish disease model of human RNASET2-deficient cystic leukoencephalopathy displays abnormalities in early microglia.

ABSTRACTHuman infantile-onset RNASET2-deficient cystic leukoencephalopathy is a Mendelian mimic of in utero cytomegalovirus brain infection with prenatally developing inflammatory brain lesions. We used an RNASET2-deficient zebrafish model to elucidate the underlying disease mechanisms. Mutant and wild-type zebrafish larvae brain development between 2 and 5 days post fertilization (dpf) was examined by confocal live imaging in fluorescent reporter lines of the major types of brain cells. In contrast to wild-type brains, RNASET2-deficient larvae displayed increased numbers of microglia with altered morphology, often containing inclusions of neurons. Furthermore, lysosomes within distinct populations of the myeloid cell lineage including microglia showed increased lysosomal staining. Neurons and oligodendrocyte precursor cells remained unaffected. This study provides a first look into the prenatal onset pathomechanisms of human RNASET2-deficient leukoencephalopathy, linking this inborn lysosomal disease to the innate immune system and other immune-related childhood encephalopathies like Aicardi-Goutières syndrome (AGS).

The cellular pathology of lysosomal diseases

With a constitutive recycling function and the capacity to digest exogenous material as well as endogenous organelles in the process of autophagy, lysosomes are at the heart of the living cell. Dynamic interactions with other cellular components ensure that the lysosomal compartment is a central point of convergence in countless diverse diseases. Inborn lysosomal (storage) diseases represent about 70 genetically distinct conditions, with a combined birth frequency of about 1 in 7500. Many are associated with macromolecular storage, causing physical disruption of the organelle and cognate structures; in neurons, ectopic dendritogenesis and axonal swelling due to distension with membraneous tubules and autophagic vacuoles are observed. Disordered autophagy is almost universal in lysosomal diseases but biochemical injury due to toxic metabolites such as lysosphingolipid molecules, abnormal calcium homeostasis and endoplasmic reticulum stress responses and immune-inflammatory processes occur. However, in no case have the mechanistic links between individual clinico-pathological manifestations and the underlying molecular defect been precisely defined. With access to the external fluid-phase and intracellular trafficking pathways, the lysosome and its diseases are a focus of pioneering investment in biotechnology; this has generated innovative orphan drugs and, in the case of Gaucher's disease, effective treatment for the haematological and visceral manifestations. Given that two-thirds of lysosomal diseases have potentially devastating consequences in the nervous system, future therapeutic research will require an integrative understanding of the unitary steps in their neuro pathogenesis. Informative genetic variants illustrated by patients with primary defects in this organelle offer unique insights into the central role of lysosomes in human health and disease. We provide a conspectus of inborn lysosomal diseases and their pathobiology; the cryptic evolution of events leading to irreversible changes may be dissociated from the cellular storage phenotype, as revealed by the outcome of therapeutic gene transfer undertaken at different stages of disease.

Serendipity: How The Concept of Inborn Lysosomal Disease Was Discovered

Serendipity: How The Concept of Inborn Lysosomal Disease Was Discovered

Serendipity: how the concept of inborn lysosomal disease was discovered.

Serendipity: how the concept of inborn lysosomal disease was discovered.

Catabolism of Asialo-GM2 in Man and Mouse: SPECIFICITY OF HUMAN/MOUSE CHIMERIC GM2 ACTIVATOR PROTEINS

Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires beta-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66-72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn(106)-Tyr(114)) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients.

Specificity of Mouse GM2 Activator Protein and β-N-Acetylhexosaminidases A and B: SIMILARITIES AND DIFFERENCES WITH THEIR HUMAN COUNTERPARTS IN THE CATABOLISM OF GM2

Tay-Sachs disease, an inborn lysosomal disease featuring a buildup of GM2 in the brain, is caused by a deficiency of beta-hexosaminidase A (Hex A) or GM2 activator. Of the two human lysosomal Hex isozymes, only Hex A, not Hex B, cleaves GM2 in the presence of GM2 activator. In contrast, mouse Hex B has been reported to be more active than Hex A in cleaving GM2 (Burg, J., Banerjee, A., Conzelmann, E., and Sandhoff, K. (1983) Hoppe Seyler's Z. Physiol. Chem. 364, 821-829). In two independent studies, mice with the targeted disruption of the Hexa gene did not display the severe buildup of brain GM2 or the concomitant abnormal behavioral manifestations seen in human Tay-Sachs patients. The results of these two studies were suggested to be attributed to the reported GM2 degrading activity of mouse Hex B. To clarify the specificity of mouse Hex A and Hex B and to better understand the observed results of the mouse model of Tay-Sachs disease, we have purified mouse liver Hex A and Hex B and also prepared the recombinant mouse GM2 activator. Contrary to the findings of Burg et al., we found that the specificities of mouse Hex A and Hex B toward the catabolism of GM2 were not different from the corresponding human Hex isozymes. Mouse Hex A, but not Hex B, hydrolyzes GM2 in the presence of GM2 activator, whereas GM2 is refractory to mouse Hex B with or without GM2 activator. Importantly, we found that, in contrast to human GM2 activator, mouse GM2 activator could effectively stimulate the hydrolysis of GA2 by mouse Hex A and to a much lesser extent also by Hex B. These results provide clear evidence on the existence of an alternative pathway for GM2 catabolism in mice by converting GM2 to GA2 and subsequently to lactosylceramide. They also provide the explanation for the lack of excessive GM2 accumulation in the Hexa gene-disrupted mice.

Bovine GM1 gangliosidosis: An inborn lysosomal disease

The results of studies on GM1 gangliosidosis of Friesian calves are reviewed in the light of present knowledge about inborn lysosomal diseases. Affected calves have poor growth rates and develop signs of incoordination and blindness at about three months of age. The disease is progressive resulting in death at six to nine months of age. Storage of GM1 ganglioside in neurons is associated with reduced activity of tissue β-galactosidase. Evidence is presented suggesting an autosomal recessive mode of inheritance. The implications of these findings relative to diagnosis and control are discussed.

GLYCOSIDASES IN HUMAN SKIN FIBROBLAST CULTURES

Five glycosidases, alpha-fucosidase, alpha-galactosidase, alpha-glucosidase, beta-mannosidase and N-acetyl-alpha-glucosaminidase were studied in human skin fibroblast cultures. The pH-dependency, kinetic properties of the enzymes and results of isoelectric focusing and ion exchange chromatography are presented. Techniques suitable for diagnosing inborn lysosomal diseases in skin fibroblast cultures are defined.

Properties of five acid hydrolases in human skin fibroblast cultures. Possible use in the diagnosis of inborn lysosomal diseases.

SummaryFive acid hydrolases, β‐galactosidase, β‐gluco‐sidase, a‐mannosidase, N‐acetyl‐β‐glucosamini‐dase, and β‐glucuronidase, were studied in human skin fibroblast cultures. The pH‐de‐pendency and kinetic properties of the enzymes as well as results of gel filtration and isoelectric focusing indicate that these enzymes are the same in fibroblasts as in liver.Techniques were defined with the aim of finding methods suitable for the diagnosis of inborn lysosomal diseases. It is stressed that great caution must be executed in the interpretation of results, since isolated enzyme deficiencies may appear in early, but not in later subcultures. Also, there were great variations in the activity levels between different subcultures.

Artificial substrates in the assay of acid glycosidases

The properties of several lysosomal acid glycosidases were studied with artificial substrates in human liver and other tissues. Gel filtration and isoelectric focusing were used for separation of various forms of the enzymes. The effect on these forms of chloride and various buffers as well as their pattern in a few inborn lysosomal diseases was investigated. β-Galactosidase was seen in three different forms on gel filtration and in two on isofocusing. Full total activity was obtained only when Cl − was present. The proportions between the various enzyme forms were dependent on pH and ionic strength. The same forms were seen in all tissues studied, but the relative proportions varied. Although high total values were often seen, normal patterns were found in mannosidosis, Gaucher's disease, and Tay-Sachs disease. β-Glucosidase was seen in five different forms on gel filtration and in three on isofocusing. All these forms were active also on the β-galactoside substrate. Cl − did not influence the total activity, but the gel filtration pattern could be changed by varying the ionic strength, but not pH. The same forms were seen in all tissues studied, but the proportions were very varying. Normal patterns were seen in mannosidosis and Tay-Sachs disease. In Hurler's disease β-glucosidase transformed more easily than in controls to large-molecular size forms, especially at low ionic strength. α-Mannosidase seemed homogeneous on gel filtration. Isofocusing demonstrated the presence of one very broad peak, indicating polydispersity. The activity was somewhat influenced by pH, but not by ionic strength when gelfiltrated. Polydispersity was noted in many tissues but the isofocusing pattern varied. The same findings as in controls were obtained in Hurler's, Hunter's, Gaucher's, and Tay-Sachs disease. N-Acetyl-β-glucosaminidase and N-acetyl-β-galactosaminidase were identical and homogeneous on gel filtration. Varying pH, buffer, and ionic strength did not change the gel filtration pattern. Isofocusing pattern showed two forms A and B. These forms were found in all tissues studied, but the B form was only 10–20% of the total activity in urine and plasma. No transformation from one form to the other was noted. Normal pattern was seen in mannosidosis, Hurler's, Hunter's, and Gaucher's disease. β-Glucuronidase was homogeneous on gel filtration but the elution volume was strongly dependent on ionic strength and pH. On isofocusing two forms were seen at the same pH (5 and 7) as hexosaminidase A and B. Very variable results were obtained with different tissues, maximum activity seen at pH 4 and pH 7 for urine and at pH 5 and 5.8 for plasma. No pathological properties, except for total activity, were found in mannosidosis, Hurler's, Hunter's, Gaucher's or Tay-Sachs disease.

Urinary excretion of acid hydrolases in disease.

Methods were devised for the assay in urine of four acid hydrolases, β-galactosidase, α-mannosidase, N-acetyl-β-glucosaminidase, and -glucuronidase. Highest activities were obtained by assay directly on centrifuged urine. In gel filtration and isoelectric focusing experiments, these enzymes had properties in the urine very similar to those in the kidneys, with the exception of N-acetyl-β-glucosaminidase B, which had only a low activity in the urine. The following findings support the theory that the enzymes derive from the kidneys and not plasma: In mucopolysaccharidosis and pregnancy, levels are high in plasma but low or normal in urine: the relation between the activity levels of the various enzymes is similar in kidney and urine, but not in plasma. The activities of these enzymes were studied in various disorders, including a few inborn lysosomal diseases. A deficiency of β-galactosidase could be demonstrated in mucopolysaccharidosis types II and III, but not type IV. To detect this deficiency, the act...

Enzymatic and biochemical diagnosis of inborn lysosomal diseases with neurological symptoms.

Inborn lysosomal diseases with neurological symptoms are reviewed with special reference to the present possibilities of establishing a diagnosis with the help of enzymatic and biochemical methods in

Two male siblings' cases of specific lipidosis found by electron microscopy.

Biopsy materials obtained from cervical lymph nodes and liver tissues of two male siblings, 5 years and 10 months old and 1 year and 11 months old, were studied electron microscopically. The results were summarized as follows: 1) Marked accumulation of lipid droplets, probably neutral fat, and cholesterol clefts in reticulum cells of lymph node, Kupffer cells and hepatic cells was the most prominent feature in these biopsies. 2) These lipids were found in both lysosomes and cytoplasmic matrices. Many of the lysosomes were considered to be autolysosomes or autophagosomes, because they contained fibrillar components, endoplasmic reticulum, glycogen particles and degraded organelles. It was suggested that these lipids might have been synthesized in lysosomes. 3) This disorder seems not to be included in the category of inborn lysosomal disease, and is considered as an unclassified one.

Deficiency of acid esterase activity in Wolman's disease.

Liver, spleen, and leucocytes from patients with acid triglyceride lipase deficiency (Wolman9s disease and its clinical variants) were also found to possess greatly reduced activity of an acid esterase acting on fatty acid esters of p-nitrophenol, thereby substantiating the view that a single enzyme is responsible for these different activities. The acid esterase was resistant to the microsomal esterase inhibitor, E600, and showed broad specificity with respect to fatty acid chain length of the p-nitrophenyl esters. Other lysosomal hydrolase activities were increased non-specifically in liver from patients, thus providing further support for the classification of acid lipase deficiency as an inborn lysosomal disease. The highly sensitive leucocyte assay provides a convenient method for the diagnosis of clinical variants of Wolman9s disease; it might therefore prove particularly useful in the early detection of affected infants, and also possibly in the differentiation of heterozygotes.