Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.”     

Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as "autophagy disorders."     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Autophagy in astrocytes: A novel culprit in lysosomal storage disorders

Neurodegeneration is a prominent feature of lysosomal storage disorders (LSDs). Emerging data identify autophagy dysfunction in neurons as a major component of the phenotype. However, the autophagy pathway in the CNS has been studied predominantly in neurons, whereas in other cell types it has been largely unexplored. We studied the lysosome-autophagic pathway in astrocytes from a murine model of multiple sulfatase deficiency (MSD), a severe form of LSD. Similar to what was observed in neurons, we found that lysosomal storage in astrocytes impairs autophagosome maturation and this, in turn, has an impact upon the survival of cortical neurons and accounts for some of the neurological features found in MSD. Thus, our data indicate that lysosomal/autophagic dysfunction in astrocytes is an important component of neurodegeneration in LSDs.     

Culture conditions found to minimize false positive diagnosis of lysosomal storage disorders

Summary The effect of culture conditions on the ultrastructure and enzyme activities of cultured skin fibroblast cells relevant to the diagnosis of lysosomal storage disorders are reported. The parameters examined were: pH of the culture media, type of media, increasing cell passage, and day of harvest. Ultrastructural changes were defined in terms of the number of lysosome-like inclusion bodies per cell according to a method devised in our laboratory and proven reliable in the detection of affected individuals. Our biochemical results included determination of enzyme activities of β-hexosaminidase, α-mannosidase, β-glucuronidase-lysosmal enzymes, arylsulfatase C, a microsomal marker, and 5' nucleotidase, a plasma membrane marker. Our results indicate that the cellular ultrastructure is more sensitive than enzyme activity to changes in culture conditions. The resulting ultrastructural “artifacts” observed under certain conditions were severe enough to result in a mistaken diagnosis. Due to certain difficulties we had previously encountered in heterozygote cultures (for lysosmal storage disorders) of amniotic cells, we decided to examine heterozygote cultures of skin fibroblasts. From these (preliminary) studies it seems that an evaluation in the pH over the pysiologic levels in the culture media may help to define between normal individuals and affected heterozygotes. On the basis of our results, we recommend that to minimize false positive ultrastructural results for the diagnosis of lysosomal storage disorders, cultures be grown in minimal essential medium, the pH of the medium carefully monitored to remain below 7.4, examining the cultures not later than cell Passage 8 and no later than Day 10 after subculture. This work was part of the requirement for the fulfillment of a Ph.D. thesis (J. A.) submitted to the Hebrew University. This work was supported in part by the Richard Meyer Fund for teratological research.     

Use of nonviral promoters in adenovirus-mediated gene therapy: reduction of lysosomal storage in the aspartylglucosaminuria mouse

BACKGROUND: Aspartylglucosaminuria (AGU) is a lysosomal storage disease with severe neurodegenerative clinical features resulting from the deficiency of lysosomal aspartylglucosaminidase (AGA). The AGU knockout mouse is a good model to test different therapy strategies, as it mimics well the human pathogenesis of the disease exhibiting storage vacuoles in all tissues. In this study we investigated the efficiency of nonviral promoters in adenovirus-mediated gene therapy. METHODS: The deficient corrective enzyme, AGA, was expressed using two tissue-specific promoters, neuron-specific enolase (NSE), astrocyte-specific (GFAP) and the endogenous AGA promoter. An intrastriatal injection site was chosen due to its wide connections in the central nervous system (CNS). The expression of AGA was analyzed 1 week, 2 weeks, 4 weeks, 2 months and 4 months after the virus injection by lysosomal AGA-specific immunostaining. A correction of the lysosomal storage in the brain of treated mice was also studied using toluidine blue stained thin sections. RESULTS: The overexpressed AGA enzyme was detected in addition to the injection site, also in the ipsilateral parietal cortex indicating migration of AGA in the brain tissue. Duration of AGA expression was markedly longer with all the viruses used compared to the green fluorescent protein (GFP) expression driven by the viral cytomegalovirus (CMV) promoter. In most animals the storage was decreased by at least 50% as compared to untreated AGU mouse brains. Remarkably, >90% correction of storage at the ipsilateral cortex was found with the NSE promoter at 4 weeks and 2 months after injection. Additionally, partial clearance of storage was demonstrated also in the contralateral side of the brain. CONCLUSIONS: These data implicate that tissue-specific promoters are especially useful in virus-mediated gene therapy aiming at long-term gene expression.     

The ultrastructure of dentinogenesis and amelogenesis in rat molar tooth germs grown as organ cultures in vitro

Summary Tooth germs from foetal rats of 17 days post-insemination were maintained in vitro for 12 days. Odontoblasts and ameloblasts differentiated and secreted their respective matrices in which mineralization occurred. The ultrastructure of the cells was qualitatively similar to that observed in normal development. Odontoblasts contained more lysosome-like bodies and were found to degenerate in some sites. Mantle dentine was formed but few von Korff fibres were observed. Calcospherites were rarely seen and the mineralizing front of dentine was predominantly linear, associated with numerous small early foci of mineral formation. Enamel showed prism formation associated with the Tomes' process of the ameloblast but some local disturbances in the pattern of enamel formation were observed.Work at the Strangeways Research Laboratory was carried out under the supervision of Dame Honor Fell, F.R.S. with the support of the Medical Research Council.     

The HOX genes are expressed, in vivo, in human tooth germs: in vitro cAMP exposure of dental pulp cells results in parallel HOX network activation and neuronal differentiation

Homeobox-containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat-poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal-like phenotype in human primary ectomesenchymal dental pulp cells.     

受体介导内吞对巨噬细胞膜电位、胞浆和溶酶体pH的影响

本文利用荧光标记方法测定了刀豆素Ａ、麦芽凝集素、酵母多糖刺激引起的巨噬细胞膜电位、胞浆ｐＨ溶酶体ｐＨ的变化。结果显示三种配体均导致细胞膜电位超极化,胞浆ｐＨ降低、溶酶体ｐＨ或高,三个生理参量趋于稳定时间稍有不同。胞浆ｐＨ的降低可能有抑制内吞的作用,溶酶体ｐＨ上升是触发溶酶体内容物外排的基本因素。内吞引起的这些变化是细胞代谢过程中自我调节和保护的表现。     

Effects of protein deficiency on selective elicitation of lysosomal enzymes in rat peritoneal macrophages

Young albino rats were fedad libitum 4, 8 or 20 % (control) protein diet for 1–4 weeks. Total activities of some of the lysosomal enzymes, namely, acid phosphatase, aryl sulphatase, Β-glucuronidase and cathepsin D, were determined in resident and protease-peptone elicited peritoneal macrophages. Total cell number, protein content and the lysosomal enzyme activities were increased significantly in protease-peptone elicited macrophages; though at a lower rate in 4 % protein-fed group compared to control ones. However, the rate of induction of the tested hydrolases was selective and their response to the stimulant varied widely. Similarly, response of each enzyme to low protein diet also varied. Thus, at 4 weeks, cathepsin D and Β-glucuronidase activities, expressed per total number of elicited macrophages were reduced by 45 and 60 %, respectively, in 4 % protein-fed animals. These results indicate that the metabolic events related to lysosomal function in macrophages, are affected by dietary restriction of proteins     

Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.