Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.     

Inheritance of Lysosomal Acid β-Galactosidase Activity and Gangliosides in Crosses of DBA/2J and Knockout Mice

GM1 gangliosidosis is a progressive neurodegenerative disease caused by deficiencies in lysosomal acid beta-galactosidase (beta-gal) and involves accumulation and storage of ganglioside GM1 and its asialo form (GA1) in brain and visceral tissues. Similar to the infantile/juvenile human disease forms, B6/129Sv beta-gal knockout (ko) mice express residual tissue beta-gal activity and significant elevations of brain GM1, GA1, and total gangliosides. Previous studies suggested that inbred DBA/2J (D2) mice may model a mild form of the human disease since total brain ganglioside and GM1 concentration is higher while beta-gal specific activity is lower (by 70-80%) in D2 mice than in inbred C57BL/6J (B6) mice and other mouse strains. A developmental genetic analysis was conducted to determine if the genes encoding beta-gal (Bgl) in the D2 and the ko mice were functionally allelic and if the reduced brain beta-gal activity in D2 mice could account for elevations in total brain gangliosides and GM1. Crosses were made between D2 mice homozygous for the Bgld allele (d/d), and either B6/129Sv mice heterozygous for the Bgl+ allele (+/-) or homozygous for the ko Bgl- allele (-/-) to generate d/+ and d/- mice. Specific beta-gal activity (nmol/mg protein/h) showed additive inheritance in brain, liver, and kidney at juvenile (21 days) and adult (255 days) ages with the d/- mice having only about 16% of the beta-gal activity as that in the +/+ mice. These results indicate that the Bgl genes in the D2 and the ko mice are noncomplementing functional alleles. However, the d/- mice did not express GA1 and had total brain ganglioside and GM1 concentrations similar to those in the d/+ and +/+ mice. These results suggest that the reduced brain beta-gal activity alone cannot account for the elevation of total brain gangliosides and GM1 in the D2 mice.     

β-Galactosidase-deficient mouse as an animal model for GM1-gangliosidosis

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis. This revised version was published online in November 2006 with corrections to the Cover Date.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

N-butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis

GM1 gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid beta-galactosidase (beta-gal), the enzyme that catabolyzes GM1 within lysosomes. Accumulation of GM1 and its asialo form (GA1) occurs primarily in the brain, leading to progressive neurodegeneration and brain dysfunction. Substrate reduction therapy aims to decrease the rate of GSL biosynthesis to counterbalance the impaired rate of catabolism. The imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ) is a competitive inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in GSL biosynthesis. Neonatal C57BL/6J (B6) and beta-gal knockout (-/-) mice were injected daily from post-natal day 2 (p-2) to p-5 with either vehicle or NB-DGJ at 600 mg or 1200 mg/kg body weight. These drug concentrations significantly reduced total brain ganglioside and GM1 content in the B6 and the beta-gal (-/-) mice. Drug treatment had no significant effect on viability, body weight, brain weight, or brain water content in the B6 and beta-gal (-/-) mice. Significant elevations in neutral lipids (GA1, ceramide, and sphingomyelin) were observed in the NB-DGJ-treated beta-gal (-/-) mice, but were not associated with adverse effects. Also, NB-DGJ treatment of B6 and beta-gal (-/-) mice from p-2 to p-5 had no subsequent effect on brain ganglioside content at p-21. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM1 content at early neonatal ages. These findings suggest that substrate reduction therapy using NB-DGJ may be an effective early intervention for GM1 gangliosidosis and possibly other GSL lysosomal storage diseases.     

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.     

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

Lysosomal accumulation of Trk protein in brain of GM₁ -gangliosidosis mouse and its restoration by chemical chaperone

G(M1) -gangliosidosis is a fatal neurodegenerative disorder caused by deficiency of lysosomal acid β-galactosidase (β-gal). Accumulation of its substrate ganglioside G(M1) (G(M1) ) in lysosomes and other parts of the cell leads to progressive neurodegeneration, but underlying mechanisms remain unclear. Previous studies demonstrated an essential role for interaction of G(M1) with tropomyosin receptor kinase (Trk) receptors in neuronal growth, survival and differentiation. In this study we demonstrate accumulation of G(M1) in the cell-surface rafts and lysosomes of the β-gal knockout (β-gal-/-) mouse brain association with accumulation of Trk receptors and enhancement of its downstream signaling. Immunofluorescence and subcellular fractionation analysis revealed accumulation of Trk receptors in the late endosomes/lysosomes of the β-gal-/- mouse brain and their association with ubiquitin and p62. Administration of a chemical chaperone to β-gal-/- mouse expressing human mutant R201C protein resulted in a marked reduction of intracellular storage of G(M1) and phosphorylated Trk. These findings indicate that G(M1) accumulation in rafts causes activation of Trk signaling, which may participate in the pathogenesis of G(M1) -gangliosidosis.     

Activation of ER stress and apoptosis by hydrogen peroxide in HeLa cells: protective role of mild heat preconditioning at 40°C

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40°C) can alter induction of ER-mediated stress and apoptosis by H(2)O(2) in HeLa cells. Protein expression of PERK, p-PERK, eIF2α and p-eIF2α was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2α branch of the UPR. A short exposure (15 min) of cells to H(2)O(2) (15-50 μM) activated the UPR: expression of p-PERK, p-eIF2α and p-IRE1α increased, and ATF6 cleavage occurred. Longer exposure (1-3h) to H(2)O(2) induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H(2)O(2). In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H(2)O(2)-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H(2)O(2)-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H(2)O(2). Moreover, the adaptive response (UPR) dominates during milder H(2)O(2) stress, whereas ER-mediated apoptosis occurs during more severe stress.     

Processing of human beta-galactosidase in GM1-gangliosidosis and Morquio B syndrome

The nature of the molecular defect resulting in the beta-galactosidase deficiency in different forms of GM1-gangliosidosis and mucopolysaccharidosis IV B (Morquio B syndrome) was investigated. Normal and mutant cultured skin fibroblasts were labeled in vivo with [3H]leucine and immunoprecipitation studies with human anti-beta-galactosidase antiserum were performed, followed by polyacrylamide gel electrophoresis and fluorography. In Morquio B syndrome, the mutation does not interfere with the normal processing and intralysosomal aggregation of beta-galactosidase. In cells from infantile and adult GM1-gangliosidosis, 85-kDa precursor beta-galactosidase was found to be synthesized normally but more than 90% of the enzyme was subsequently degraded at one of the early steps in posttranslational processing. The residual 5-10% beta-galactosidase activity in adult GM1-gangliosidosis is 64-kDa mature lysosomal enzyme with normal catalytic properties but with a reduced ability of the monomeric form to aggregate into high molecular weight multimers. Knowledge of the exact nature of the molecular defect underlying beta-galactosidase deficiency in man may lead to a better understanding of the clinical and pathological heterogeneity among patients with different types of GM1-gangliosidosis and Morquio B syndrome.     

AAV-mediated gene delivery in adult GM1-gangliosidosis mice corrects lysosomal storage in CNS and improves survival

Background

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid β-galactosidase (βgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis.

Methodology/Principal Findings

In this study we examined the effect of thalamic infusion of AAV2/1-βgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice.

Conclusions/Significance

Our studies show that the AAV-modified thalamus can be used as a ‘built-in’ central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice.     

Treadmill exercise represses neuronal cell death and inflammation during Aβ-induced ER stress by regulating unfolded protein response in aged presenilin 2 mutant mice

Alzheimer’s disease (AD) is characterized by the deposition of aggregated amyloid-beta (Aβ), which triggers a cellular stress response called the unfolded protein response (UPR). The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER) stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of neuronal apoptosis and inflammation by Aβ-induced ER stress to exercise training are not fully understood. Here, we demonstrated that treadmill exercise (TE) prevented PS2 mutation-induced memory impairment and reduced Aβ-42 deposition through the inhibition of β-secretase (BACE-1) and its product, C-99 in cortex and/or hippocampus of aged PS2 mutant mice. We also found that TE down-regulated the expression of GRP78/Bip and PDI proteins and inhibited activation of PERK, eIF2α, ATF6α, sXBP1 and JNK-p38 MAPK as well as activation of CHOP, caspase-12 and caspase-3. Moreover, TE up-regulated the expression of Bcl-2 and down-regulated the expressions of Bax in the hippocampus of aged PS2 mutant mice. Finally, the generation of TNFα and IL-1α and the number of TUNEL-positive cells in the hippocampus of aged PS2 mutant mice was also prevented or decreased by TE. These results showed that TE suppressed the activation of UPR signaling pathways as well as inhibited the apoptotic pathways of the UPR and inflammatory response following Aβ-induced ER stress. Thus, therapeutic strategies that modulate Aβ-induced ER stress through TE could represent a promising approach for the prevention or treatment of AD.     

Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.     

The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages

Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop-/- macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages.