VMA21 deficiency causes an autophagic myopathy by compromising V-ATPase activity and lysosomal acidification.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

Liquid chromatography/electrospray ionisation-tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma.

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted ß-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.

Synthesis of 1,5-dideoxy-1,5-iminoribitol C-glycosides through a nitrone-olefin cycloaddition domino strategy: identification of pharmacological chaperones of mutant human lysosomal ß-galactosidase.

We report herein a newly developed domino reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-iminoribitol iminosugar C-glycosides 7a-e and 8. The key intermediate in this approach is a six-membered cyclic sugar nitrone that is generated in situ and trapped by an alkene dipolarophile via a [2 + 3] cycloaddition reaction to give the corresponding isooxazolidines 10a-e in a "one-pot" protocol. The iminoribitol C-glycosides 7a-e and 8 were found to be modest ß-galactosidase (bGal) inhibitors. However, compounds 7c and 7e showed "pharmacological chaperone" activity for mutant lysosomal bGal activity and facilitated its recovery in GM1 gangliosidosis patient fibroblasts by 2-6-fold.

Juvenile-onset motor neuron disease caused by novel mutations in ß-hexosaminidase.

A 12 year-old female presented with a seven-year history of progressive muscle weakness, atrophy, tremor and fasciculations. Cognition was normal. Rectal biopsy revealed intracellular storage material and biochemical testing indicated low hexosaminidase activity consistent with juvenile-onset G(M2)-gangliosidosis. Genetic evaluation revealed compound heterozygosity with two novel mutations in the hexosaminidase ß-subunit (c.512-3 C>A and c.1613+15_1613+18dup). Protein analysis was consistent with biochemical findings and indicated only a small portion of ß-subunits were properly processed. These results provide additional insight into juvenile-onset G(M2)-gangliosidoses and further expand the number of ß-hexosaminidase mutations associated with motor neuron disease.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant ß-glucocerebrosidase in plant cells: evidence that N370S is a folding mutant.

Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid ß-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81-95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER.

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy.

Deficiencies of lysosomal ß-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant ß-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal ß-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.

An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants).

Late-onset GM2 gangliosidosis is an autosomal recessive, neurodegenerative, lysosomal storage disease, caused by deficiency of ß-hexosaminidase A (Hex A), resulting from mutations in the HEXA (Tay-Sachs variant) or the HEXB (Sandhoff variant) genes. The enzyme deficiency in many patients with juvenile or adult onset forms of the disease results from the production of an unstable protein, which becomes targeted for premature degradation by the quality control system of the smooth endoplasmic reticulum and is not transported to lysosomes. In vitro studies have shown that many mutations in either the α or ß subunit of Hex A can be partially rescued, i.e. enhanced levels of both enzyme protein and activity in lysosomes, following the growth of patient cells in the presence of the drug, pyrimethamine. The objectives of the present clinical trial were to establish the tolerability and efficacy of the treatment of late-onset GM2 gangliosidosis patients with escalating doses of pyrimethamine, to a maximum of 100 mg per day, administered orally in a single daily dose, over a 16-week period . The primary objective, tolerability, was assessed by regular clinical examinations, along with a panel of hematologic and biochemical studies. Although clinical efficacy could not be assessed in this short trial, treatment efficacy was evaluated by repeated measurements of leukocyte Hex A activity, expressed relative to the activity of lysosomal ß-glucuronidase. A total of 11 patients were enrolled, 8 males and 3 females, aged 23 to 50 years. One subject failed the initial screen, another was omitted from analysis because of the large number of protocol violations, and a third was withdrawn very early as a result of adverse events which were not drug-related. For the remaining 8 subjects, up to a 4-fold enhancement of Hex A activity at doses of 50 mg per day or less was observed. Additionally marked individual variations in the pharmacokinetics of the drug among the patients were noted. However, the study also found that significant side effects were experienced by most patients at or above 75 mg pyrimethamine per day. We concluded that pyrimethamine treatment enhances leukocyte Hex A activity in patients with late-onset GM2 gangliosidosis at doses lower than those associated with unacceptable side effects. Further plans are underway to extend these trials and to develop methods to assess clinical efficacy.

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Fluorous iminoalditols: a new family of glycosidase inhibitors and pharmacological chaperones.

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D-galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D-galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.

Identification of proteins in the ceroid-like autofluorescent aggregates from liver lysosomes of Beige, a mouse model for human Chediak-Higashi syndrome.

Chediak-Higashi syndrome is characterized by oculocutaneous albinism, a bleeding tendency and severe recurrent infections. Age-dependent formations of autofluorescent ceroid-like substances have been noted in a variety of tissues. In this study, we isolated an autofluorescent ceroid-like aggregate from purified Beige mouse liver lysosomes and analyzed the composition of the aggregate by ion trap mass-spectrometry. In addition to lysosomal proteins, this aggregate contains proteins normally localized in the ER, mitochondria, peroxisomes, and the cytosol. Bip, a luminal ER protein was abundant in lysosomal ceroid. The ER, mitochondria, and cytosol proteins could arise in lysosomes through stimulation of autophagy, but we found no differences between normal and CHS fibroblasts in the degree of lysosomal acidity and in the level of conversion of soluble microtubular-associated protein 1 light chain 3 type I to membrane-associated type II, an accepted probe for hyper-autophagy suggesting that ceroid formation is unlikely to arise via this mechanism.

Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors.

N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C 6alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against ß-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related ß-galactosidase mutants.

Isofagomine induced stabilization of glucocerebrosidase.

Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis.

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression.

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.

Purification and proteomic analysis of lysosomal integral membrane proteins.

Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping membrane has received scant attention. Advances in mass spectrometry and proteomics have laid the necessary groundwork to facilitate investigation of membranes such as those of lysosomes, mitochondria, and other organelles to find novel proteins and novel functions. Pure lysosomes are a prerequisite, and we have successfully identified an abundance of membrane proteins from lysosomes of rat liver. Here, we describe two comparable and easy methods to isolate lysosomes from mouse or rat liver in sufficient quantities for proteomics studies. Also included is a comparison of the soluble, luminal proteins obtained from each of the two preparations separated by 2D immobilized pH gradient (IPG) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.

NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders.

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.