Lysosomal α-D-mannosidase

-mannosidosis in the human is an autosomal recessive lysosomal storage disease caused by a deficiency of lysosomal -D-mannosidasea actvity. Lysosomal -D-mannosidase is involved in the catabolism of N-linked glycoproteins through the sequential degradation of high-mannose, hybrid and complex oligosaccharides. This review is focused on human, mouse, bovine and feline genes coding for lysosomal -D-mannosidase. In particular the exon-intron structure of the genes, their promoters, and the identification of mutations causing the disease have been examined. The construction, by homologous recombination, of a mouse model of -mannosidosis is reported.     

Functions of the ��, ��, and �� Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α₂β₂γ₂ hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α₂β₂γ₂ and α₂β₂ forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the acid hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.     

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.     

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.     

Lysosomal localization of β-fructofuranosidase-containing liposomes injected into rats. Some implications in the treatment of genetic disorders

Yeast beta-fructofuranosidase (invertase) or (131)I-labelled albumin were entrapped into liposomes composed of phosphatidylcholine, cholesterol and phosphatidic acid. Of the beta-fructofuranosidase activity in the liposomal preparations 96-100% was latent. The following observations were made in experiments with rats injected with protein-containing liposomes. 1. After injection of beta-fructofuranosidase-containing liposomes (220 units or 1.5mg of beta-fructofuranosidase and 17.5mg of lipid), beta-fructofuranosidase activity in blood retained its latency but the activity declined to 50% of the injected dose in 1h. Within 6h much of this activity was recovered in the liver and spleen (respectively 45% and 10% of that injected). For up to 21h after injection, the mitochondrial-lysosomal fraction was the principal location of the hepatic beta-fructofuranosidase activity. 2. Lysosomal localization of liposomal protein was supported by the observed increase in the trichloroacetic acid-soluble radioactivity during incubation of the lysosome-rich fraction of the liver of rats injected with liposomes containing (131)I-labelled albumin. 3. Association of liposomal protein with lysosomes was demonstrated on subfractionation of the mitochondrial-lysosomal fraction of the liver of rats injected with beta-fructofuranosidase-containing liposomes in a Ficoll-mannitol gradient. beta-Fructofuranosidase, lysosomal and mitochondrial enzyme marker activities were found to exhibit similar distribution patterns along the gradient. However, in similar experiments with rats previously injected with Triton WR-1339 or dextran (known to alter the specific gravity of lysosomes), only beta-fructofuranosidase and lysosomal marker moved along the gradient, in strikingly similar patterns. 4. The lysosomal localization of injected liposome-entrapped material can probably be utilized in the treatment of certain disorders in man.     

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.     

Loss of β-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells

To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson’s disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD.     

Biological Time-Related Changes in Tolerance of Male Mice to Hypoxia—II. Orcadian Rhythm of Lysosomal Susceptibility to Hypoxia

Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of β-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum β-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes.     

Neurologic Abnormalities in Mouse Models of the Lysosomal Storage Disorders Mucolipidosis II and Mucolipidosis III γ

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α₂β₂γ₂ hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.     

Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-β (Aβ), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aβ is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aβ generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aβ generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.     

Guanosine 5′-[γ-thio]triphosphate-Mediated Activation of Cytosol Phospholipase C Caused Lysosomal Destabilization

Lysosomal disintegration is critical for the organelle functions and cellular viability. In this study, we established that guanosine 5′-[γ-thio]triphosphate (GTP-γ-S)-activated cytosol of rat hepatocytes could increase lysosomal permeability to both potassium ions and protons and osmotically destabilize the lysosomes via K⁺/H⁺ exchange. These results were obtained through measurements of lysosomal β-hexosaminidase-free activity, membrane potential and intralysosomal pH. Assays of phospholipase C (PLC) activity show that cytosolic PLC was activated upon addition of GTP-γ-S to the cytosol. The effects of cytosol on the lysosomes could be abolished by D609, an inhibitor of PLC, but not by the inhibitors of phospholipase A₂. The cytosol-treated lysosomes disintegrated markedly in hypotonic sucrose medium, reflecting that the lysosomal osmotic sensitivity increased. Microscopic observations showed that the lysosomes became more swollen in hypotonic sucrose medium. This indicates that the cytosol treatment induced osmotic shock to the lysosomes and an influx of water into the organelle. Xiang Wang and Li-Li Wang contributed equally to this work.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Development of targeted therapies for Parkinson’s disease and related synucleinopathies

Therapeutic efforts in neurodegenerative diseases have been very challenging, particularly due to a lack of validated and mechanism-based therapeutic targets and biomarkers. The basic idea underlying the novel therapeutic approaches reviewed here is that by exploring the molecular basis of neurodegeneration in a rare lysosomal disease such as Gaucher’s disease (GD), new molecular targets will be identified for therapeutic development in common synucleinopathies. Accumulation of α-synuclein plays a key role in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies, suggesting that improved clearance of α-synuclein may be of therapeutic benefit. To achieve this goal, it is important to identify specific mechanisms and targets involved in the clearance of α-synuclein. Recent discovery of clinical, genetic, and pathological linkage between GD and PD offers a unique opportunity to examine lysosomal glucocerebrosidase, an enzyme mutated in GD, for development of targeted therapies in synucleinopathies. While modulation of glucocerebrosidase and glycolipid metabolism offers a viable approach to treating disorders associated with synuclein accumulation, the compounds described to date either lack the ability to penetrate the CNS or have off-target effects that may counteract or limit their capabilities to mediate the desired pharmacological action. However, recent emergence of selective inhibitors of glycosphingolipid biosynthesis and noninhibitory pharmacological chaperones of glycosphingolipid processing enzymes that gain access to the CNS provide a novel approach that may overcome some of the limitations of compounds reported to date. These new strategies may allow for development of targeted treatments for synucleinopathies that affect both children and adults.     

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.     

Lysosomal Storage Diseases: Is Impaired Apoptosis a Pathogenic Mechanism?

Lysosomal storage disorders are inborn diseases resulting from the lack or activity of lysosomal hydrolases, transporters, or integral membrane proteins. Although most of the genes encoding these proteins have been characterized and many gene defects identified, the molecular bases underlying the pathophysiology of these genetic diseases still remain obscure. In this mini-review, the potential role of apoptotic cell death in the development of the cellular and tissue lesions seen in lysosomal storage disorders, and particularly in neurological diseases, is discussed. A list of observations documenting either a decrease or an exacerbation in apoptosis induction are presented. The putative, yet controversial contribution of certain sphingolipids and cathepsins in the regulation of these phenomena is emphasized.     

Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress,Dysfunction of Calcium Signalling,and Inhibition of Autophagy–Lysosomal Pathway

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-d-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic–lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.     

Lysosomal storage disorder in non-immunological hydrops fetalis (NIHF) - more common than assumed? Report of four cases with transient NIHF and a review of the literature

Background

Lysosomal storage disorders (LSD) are a rare cause of non immunological hydrops fetalis (NIHF) and congenital ascites. The reported incidence is about 1%. The incidence of idiopathic NIHF is estimated to be about 18%.

Patients and methods

We report four cases with transient hydrops fetalis resulting from LSD and performed a literature review on LSD with NIHF and congenital ascites in combination.

Results

At present, 12 different LSDs are described to be associated with NIHF or congenital ascites. Most patients had a family history of NIHF, where the preceding sibling had not been examined. A diagnostic approach to the fetus with NIHF due to suspected LSD either in utero or postnatal is suggested. Transient forms of NIHF and/or ascites in association with MPS IVA, MPS VII and NPC are described for the first time in this publication.

Conclusions

LSD should be considered in transient hydrops. Enzymatic studies in chorionic villous sample or amniotic cultured cells, once the most common conditions associated with fetal ascites or hydrops have been ruled out, are important. This paper emphasizes the fact that LSD is significantly higher than the estimated 1% in previous studies, which is important for genetic counseling as there is a high risk of recurrence and the availability of enzyme replacement therapy for an increasing number of LSD.