Bis(monoacylglycero)phosphate: a secondary storage lipid in the gangliosidoses

Bis(monoacylglycero)phosphate (BMP) is a negatively charged glycerophospholipid with an unusual sn-1;sn-1′ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. Elevated BMP levels have been found in many lysosomal storage diseases (LSDs), suggesting an association with lysosomal storage material. The gangliosidoses are a group of neurodegenerative LSDs involving the accumulation of either GM1 or GM2 gangliosides resulting from inherited deficiencies in β-galactosidase or β-hexosaminidase, respectively. Little information is available on BMP levels in gangliosidosis brain tissue. Our results showed that the content of BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. The storage of BMP and ganglioside GM2 in brain were reduced similarly following adeno-associated viral-mediated gene therapy in Sandhoff disease mice. We also found that C22:6, C18:0, and C18:1 were the predominant BMP fatty acid species in gangliosidosis brains. The results show that BMP accumulates as a secondary storage material in the brain of a broad range of mammals with gangliosidoses.     

Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+-ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis

Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24-fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3-fold increased cholesterol content, and a 1.4-fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+,K+-ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+,K+-ATPase.     

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd−/− and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd−/− and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd−/− mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd−/− mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd−/− mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.     

Characterization of candidate genes for neuronal ceroid lipofuscinosis in dog

The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of monogenic autosomal recessive inherited progressive neurodegenerative diseases characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Until today, eight forms of NCL have been classified in humans by clinical criteria, which result from mutations in at least six different genes (TPP1, CLN2, PPT1, CLN5, CLN6, and CLN8). NCL has also been reported in various domestic animal species including cattle, goat, sheep, cat, and certain dog breeds. In this report, the experimental analysis of canine PPT1, CLN5, CLN6, and CLN8 full-length cDNA sequences is described, and the current whole genome sequence assembly was used for gene structure analyses. Characterization of the four canine genes revealed a conserved organization with respect to the human orthologs. In general the gene size in dog is smaller compared to the human sequence due to shorter intron length. Using four individuals of Tibetan terrier with NCL, and a single affected Polish Owczarek Nizinny (PON) dog, we excluded the complete coding region of canine PPT1 and CLN8 and three of four exons of CLN5 and six of seven exons of CLN6 harboring disease-causing mutations.     

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.     

Accumulation of Lysosphingolipids in Tissues from Patients with GM1 and GM2 Gangliosidoses

By using a sensitive method, we assayed lysocompounds of gangliosides and asialogangliosides in tissues from four patients with GM2 gangliosidosis (one with Sandhoff disease and three with Tay-Sachs disease) and from three patients with GM1 gangliosidosis [one with infantile type (fetus), one with late-infantile, and one with adult type]. In the brain and spinal cord of all the patients except for an adult GM1 gangliosidosis patient, abnormal accumulation of the lipids was observed, though the concentration in the fetal tissue was low. In GM2 gangliosidosis, the amounts of lyso GM2 ganglioside accumulated in the brain were similar among the patient with Sandhoff disease and the patients with Tay-Sachs disease, whereas the concentration of asialo lyso GM2 ganglioside in the brain was higher in the former patient than in the latter patients. By comparing the sphingoid bases of neutral sphingolipids, gangliosides, and lysosphingolipids, it was suggested that lysosphingolipids in the diseased tissue are synthesized by sequential glycosylation from free sphingoid bases, but not by deacylation of the sphingolipids. Because lysosphingolipids are known to be cytotoxic, the abnormally accumulated lysophingolipids may well be the pathogenetic agent for the neuronal degeneration in gangliosidoses.     

Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.     

Building a cathedral

This article describes the scientific legacy of Dr. Leon Wolfe, with illustrations from his contributions to insect physiology, glycolipid, and eicosanoid biochemistry as well as to our understanding of neuronal ceroid lipofuscinoses and lysosomal storage diseases. In addition to the written record, Wolfe inspired all who knew him with his boundless imagination and enthusiasm for science and his ability to see the promise and potential of every experiment.     

Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells

Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal β-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of β-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum α-glucosidases and lysosomal β-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.     

From sheep to mice to cells: Tools for the study of the sphingolipidoses

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann–Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.     

Neurochemical, morphological, and neurophysiological abnormalities in retinas of Sandhoff and GM1 gangliosidosis mice

Retinal abnormalities are well documented in patients with ganglioside storage diseases. The total content and distribution of retinal glycosphingolipids was studied for the first time in control mice and in Sandhoff disease (SD) and GM1 gangliosidosis mice. Light and electron microscopy of the SD and the GM1 retinas revealed storage in ganglion cells. Similar to previous findings in rat retina, GD3 was the major ganglioside in mouse retina, while GM2 and GM1 were minor species. Total ganglioside content was 44% and 40% higher in the SD and the GM1 retinas, respectively, than in the control retinas. Furthermore, GM2 and GM1 content were 11-fold and 51-fold higher in the SD and the GM1 retinas than in the control retinas, respectively. High concentrations of asialo-GM2 and asialo-GM1 were found in the SD and the GM1 retinas, respectively, but were undetectable in the control retinas. The GSL abnormalities in the SD and the GM1 retinas reflect significant reductions in beta-hexosaminidase and beta-galactosidase enzyme activities, respectively. Although electroretinograms appeared normal in the SD and the GM1 mice, visual evoked potentials were subnormal in both mutants, indicating visual impairments. Our findings present a model system for assessing retinal pathobiology and therapies for the gangliosidoses.     

Hippocampal commissure defects in crosses of four inbred mouse strains with absent corpus callosum

It is known that four common inbred mouse strains show defects of the forebrain commissures. The BALB/cJ strain has a low frequency of abnormally small corpus callosum, whereas the 129 strains have many animals with deficient corpus callosum. The I/LnJ and BTBR T+ tf/J strains never have a corpus callosum, whereas half of I/LnJ and almost all BTBR show severely reduced size of the hippocampal commissure. Certain F1 hybrid crosses among these strains are known to be less severely abnormal than the inbred parents, suggesting that the parent strains have different genetic causes of commissure defects. In this study, all hybrid crosses among the four strains were investigated. The BTBR × I/Ln hybrid expressed almost no defects of the hippocampal commissure, unlike its inbred parent strains. Numerous three‐way crosses among the four strains yielded many mice with no corpus callosum and severely reduced hippocampal commissure, which shows that the phenotypic defect can result from several different combinations of genetic alleles. The F2 and F3 hybrid crosses of BTBR and I/LnJ had almost 100% absence of the corpus callosum but about 50% frequency of deficient hippocampal commissure. The four‐way hybrid cross among all four abnormal strains involved highly fertile parents and yielded a very wide phenotypic range of defects from almost no hippocampal commissure to totally normal forebrain commissures. The F2 and F3 crosses as well as the four‐way cross provide excellent material for studies of genetic linkage and behavioral consequences of commissure defects.     

Increased Acetylcholine Synthesis and Release in Brains of Cats with GM1 Gangliosidosis

Cholinergic processes were measured in motor cortex, hippocampus, and striatum of cats in the terminal stages of GM1 gangliosidosis and compared to those of control cats. The greatest difference observed was elevation in the rate of K+-stimulated release of acetylcholine (ACh) from brain slices prepared from affected cats. The K+-stimulated release of endogenous ACh was increased by 31-43% and of newly synthesized ACh by 19-80% in brain slices from different brain regions. All regions that were examined were affected but the greatest effects occurred in cortex. The rate of synthesis of ACh was elevated in cortical and hippocampal slices. Choline acetyltransferase activity in brain regions of cats with GM1 gangliosidosis was not significantly different from that in controls, whereas high-affinity choline transport in cortical synaptosomes was elevated. Muscarinic receptor binding sites were reduced in the cortex, hippocampus, and striatum of GM1 mutant cats, whereas the apparent affinity was not altered. These results indicate that there are major alterations of cholinergic function in the brains of cats with GM1 gangliosidosis.     

Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs).

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pH(i) values were measured by the membrane-permeant acetoxymethyl ester of the indicator dye, 2',7'-bis(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin-like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.     

Molecular forms of GM2-activator protein. A study on its biosynthesis in human skin fibroblasts

The biosynthesis and secretion of lysosomal GM2-activator was studied in fibroblasts from controls and patients of GM2 gangliosidosis metabolically labelled with [3H]-leucine. Immunoprecipitation was performed with affinity-purified antibodies to human kidney GM2-activator protein. Normal fibroblasts and fibroblasts of variant B and O of GM2 gangliosidosis secrete GM2-activator protein as a 24-kDa polypeptide, which is able to stimulate degradation of ganglioside GM2 by beta-hexosaminidase A in the in vitro assay. In the presence of 10mM NH4Cl the rate of secretion is twice as high as in normal fibroblasts. Intracellularly, GM2-activator protein is represented in these cell lines by polypeptides with apparent molecular masses ranging from 21 kDa-22.5 kDa. Under the same labelling conditions, in two cell lines of patients with variant AB of infantile GM2 gangliosidosis intracellularly only traces of GM2-activator were detectable, whereas significant amounts of polypeptides with molecular masses between 25 and 26.5 kDa could be precipitated from the media of these fibroblasts.     

Macroautophagy Is Not Directly Involved in the Metabolism of Amyloid Precursor Protein

Alterations in the metabolism of amyloid precursor protein (APP) are believed to play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate that APP is proteolytically processed in endosomal-autophagic-lysosomal compartments. In this study, we used both in vivo and in vitro paradigms to determine whether alterations in macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse models; however, discrepancies between LC3-II and APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous murine Aβ40 from RIPA-soluble extracts was increased in brains of all three mice. The in vivo relationship between AV and APP-CTF accumulation was also seen in cultured neurons treated with agents that impair primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by agents that induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive lysosomes. These data suggest that neuronal macroautophagy does not directly regulate APP metabolism but highlights the important anti-amyloidogenic role of lysosomal proteolysis in post-secretase APP-CTF catabolism.     

Association analysis between canine behavioural traits and genetic polymorphisms in the Shiba Inu breed

The relationships between behavioural trait data and the genotype of 15 polymorphisms in eight neurotransmitter-related genes were analysed in 77 dogs of the Shiba Inu breed, an indigenous Japanese dog. The data were obtained from a 26-item questionnaire on the dog's behaviour, distributed to the dog's owners, through veterinary hospitals and the Shiba Inu breed magazine. A factor analysis of the questionnaire items extracted eight factors accounting for 66.8% of the variance. An association analysis between these factors and genetic polymorphisms indicated that the polymorphism of c.471T>C in the solute carrier family 1 ( neuronal/epithelial high-affinity glutamate transporter ) member 2 ( SLC1A2 ) gene was significantly associated with Factor 1, referred to as 'aggression to strangers'. This association remained stable in separate analyses of data from surveys obtained from the hospitals and those obtained from the magazine. The results suggest that the c.471T>C polymorphism is associated with some types of aggressive behaviour in the Shiba Inu. Further studies using other dog breeds are necessary to extend these findings to dogs in general.     

Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence

Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.