Finding pathogenic commonalities between Niemann-Pick type C and other lysosomal storage disorders: Opportunities for shared therapeutic interventions

Lysosomal storage disorders (LSDs) are diseases characterized by the accumulation of macromolecules in the late endocytic system and are caused by inherited defects in genes that encode mainly lysosomal enzymes or transmembrane lysosomal proteins. Niemann-Pick type C disease (NPCD), a LSD characterized by liver damage and progressive neurodegeneration that leads to early death, is caused by mutations in the genes encoding the NPC1 or NPC2 proteins. Both proteins are involved in the transport of cholesterol from the late endosomal compartment to the rest of the cell. Loss of function of these proteins causes primary cholesterol accumulation, and secondary accumulation of other lipids, such as sphingolipids, in lysosomes. Despite years of studying the genetic and molecular bases of NPCD and related-lysosomal disorders, the pathogenic mechanisms involved in these diseases are not fully understood. In this review we will summarize the pathogenic mechanisms described for NPCD and we will discuss their relevance for other LSDs with neurological components such as Niemann- Pick type A and Gaucher diseases. We will particularly focus on the activation of signaling pathways that may be common to these three pathologies with emphasis on how the intra-lysosomal accumulation of lipids leads to pathology, specifically to neurological impairments. We will show that although the primary lipid storage defect is different in these three LSDs, there is a similar secondary accumulation of metabolites and activation of signaling pathways that can lead to common pathogenic mechanisms. This analysis might help to delineate common pathological mechanisms and therapeutic targets for lysosomal storage diseases.     

Treatments for lysosomal storage disorders

There are over 70 human diseases that are caused by defects in various aspects of lysosomal function. Until 20 years ago, the only specific therapy available for lysosomal storage disorders was allogeneic haemopoietic stem cell transplantation. Over the last two decades, there has been remarkable progress and there are now licensed treatments for seven of these diseases. In some cases, a choice of agents is available. For selected enzyme-deficiency disordes, ERT (enzyme-replacement therapy) has proved to be highly effective. In other cases, ERT has been less impressive, and it seems that it is not possible to efficiently deliver recombinant enzyme to all tissues. These difficulties have led to the development of other small-molecule-based therapies, and a drug for SRT (substrate-reduction therapy) is now licensed and potential chaperone molecules for ERT are in the late stages of clinical development. Nonetheless, there is still significant unmet clinical need, particularly when it comes to treating LSDs which affect the brain. LSDs have led the way in the development of treatment for genetic disorders, and it seems likely that there will be further therapeutic innovations in the future.     

From Lysosomal Storage Disorders to Parkinson’s Disease – Challenges and Opportunities

Lysosomes are specialized organelles with an acidic pH that act as recycling hubs for intracellular and extracellular components. They harbour numerous different hydrolytic enzymes to degrade substrates like proteins, peptides, and glycolipids. Reduced catalytic activity of lysosomal enzymes can cause the accumulation of these substrates and loss of lysosomal integrity, resulting in lysosomal dysfunction and lysosomal storage disorders (LSDs). Post-mitotic cells, such as neurons, seem to be highly sensitive to damages induced by lysosomal dysfunction, thus LSDs often manifest with neurological symptoms. Interestingly, some LSDs and Parkinson’s disease (PD) share common cellular pathomechanisms, suggesting convergence of aetiology of the two disease types. This is further underlined by genetic associations of several lysosomal genes involved in LSDs with PD. The increasing number of lysosome-associated genetic risk factors for PD makes it necessary to understand functions and interactions of lysosomal proteins/enzymes both in health and disease, thereby holding the potential to identify new therapeutic targets. In this review, we highlight genetic and mechanistic interactions between the complex lysosomal network, LSDs and PD, and elaborate on methodical challenges in lysosomal research.     

Lysosomes and lysosomal storage diseases

Lysosomal storage disorders (LSDs) are monogenic inborn errors of metabolism. Various groups have been delineated according to the affected pathway and the accumulated substrate, and new entities are still being identified. They are severe disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement, and high morbidity and mortality. Most of the genes encoding the lysosomal enzymes have been cloned, and animal models have been obtained for almost each disease. In the last decades, LSDs have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have allowed the successful development of bone marrow transplantation, substrate deprivation, enzyme replacement therapy and gene transfer methods as therapeutic options for several LSDs. The aim of this paper is to review the biology of acid hydrolases and lysosomal membrane proteins, to describe the systematic classification of LSDs and the most recently identified entities, and to briefly review novel therapeutic approaches for two lipidoses: Gaucher disease and Fabry disease.     

New therapeutic options for lysosomal storage disorders: enzyme replacement,small molecules and gene therapy

During the last few years, much progress has been made in the treatment of lysosomal storage disorders. In the past, no specific therapy was available for the affected patients, and management consisted solely of supportive care and treatment of complications. Since enzyme replacement therapy has been successfully introduced for patients with Gaucher disease, this principle of treatment has been taken into consideration for other lysosomal storage disorders as well. Clinical trials could demonstrate the clinical benefit of this therapeutic principle in Fabry disease, mucopolysaccharidoses type I, II and VI and in Pompe disease. However, the usefulness of enzyme replacement therapy is limited due to the fact that a given enzyme preparation does not have beneficial effects on all aspects of a disorder in the same degree. Additionally, clinical studies have shown that many symptoms of a lysosomal storage disorder even after long-term treatment are no more reversible. A further novel therapeutic option for lysosomal storage disorders consists of the application of small molecules that either inhibit a key enzyme which is responsible for substrate synthesis (substrate deprivation) or act as a chaperone to increase the residual activity of the lysosomal enzyme (enzyme enhancing therapy). Various gene therapeutic techniques (in vivo and ex vivo technique) have been developed in order to administer the gene that is defective in a patient to the bloodstream or directly to the brain in order to overcome the blood–brain barrier. This review will give an insight into these newly developed therapeutic strategies and will discuss their advantages and limitations.     

Gene therapy of Gaucher's and Fabry's diseases: current status and prospects

Gaucher disease and Fabry disease are lysosomal storage disorders characterized by the accumulation of sphingolipids. In both cases, the goal of gene therapy is to permanently provide tissues with enzyme levels allowing to avoid storage of the undigested substrates. Different gene therapy strategies must however be designed as Gaucher disease is due to a deficiency in the membrane-associated enzyme glucocerebrosidase, whereas Fabry disease is caused by a deficiency in the soluble enzyme alpha-galactosidase. Indeed, a soluble enzyme can be provided to tissues is trans by gene-modified cells whereas gene transfer has to target the most affected cells in the case of membrane-bound enzymes. Thus, in non-neurological Gaucher disease (type 1), the hematopoietic tissue has to be targeted as the deficiency affects the monocyte/macrophage lineage. Following promising preclinical studies, clinical protocols have been initiated to explore the feasibility and safety of retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with type 1 Gaucher disease. Although gene-marked cells were detected in vivo, the level of corrected cells was very low, a finding indicating that improved vectors along with partial myeloablation may be necessary. Here, lentiviral vectors should enable more gene transduction into the hematopoietic target cells. As concerns the diffuse neurological lesions in types 2 and 3 of Gaucher disease, they will probably be especially difficult to target by gene therapy because of the non soluble nature of glucocerebrosidase. Finally, over the last few years, Fabry disease has become a compelling target for gene therapy as an etiology-based treatment strategy. Indeed, several recent studies aiming at creating a large in vivo source of alpha-galactosidase have yielded positive long-term results in the Fabry knock-out mouse model.     

Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders

Glycosphingolipid (GSL) lysosomal storage disorders are a small but challenging group of human diseases to treat. Although these disorders appear to be monogenic in origin, where the catalytic activity of enzymes in GSL catabolism is impaired, the clinical presentation and severity of disease are heterogeneous. Present attitudes to treatment demand individual therapeutics designed to match the specific disease-related gene defect; this is an acceptable approach for those diseases with high frequency, but it lacks viability for extremely rare conditions. An alternative therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to balance cellular GSL biosynthesis with the impairment in catalytic activity seen in lysosomal storage disorders. The development of N-alkylated iminosugars that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase, offers a generic therapeutic for the treatment of all glucosphingolipidoses. The successful use of N-alkylated iminosugars to establish SRT as an alternative therapeutic strategy has been demonstrated in in vitro, in vivo and in clinical trials for type 1 Gaucher disease. The implications of these studies and the prospects of improvement to the design of iminosugar compounds for treating Gaucher and other GSL lysosomal storage disorders will be discussed.     

Non‐viral nucleic acid delivery to the central nervous system and brain tumors

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non‐viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre‐existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non‐viral gene delivery vectors.     

Cutaneous gene transfer and therapy: the present and the future

The easy accessibility of the skin as a therapeutic target provides an exciting potential for this organ for the development of gene therapy protocols for cutaneous diseases and a variety of metabolic disorders. Thus far, full phenotypic reversion of a diseased phenotype has been achieved in vivo for junctional epidermolysis bullosa and X-linked or lamellar ichthyosis and in vitro for xeroderma pigmentosum. These recessive skin diseases are characterized by skin blistering, abnormalities in epidermal differentiation and increased development of skin cancers, respectively. Corrective gene delivery at both molecular and functional levels was achieved by transduction of cultured skin cells using retroviral vectors carrying the specific curative cDNA. These positive results should prompt clinical trials based on transplantation of artificial epithelia reconstructed ex vivo using genetically modified keratinocytes. Promising results have also been obtained in phenotypic reversion of cells isolated from patients suffering from a number of metabolic diseases such as gyrate atrophy, familial hypercholesterolemia or phenylketonuria. In these diseases transplantation of autologous artificial epithelia expressing the transgenes of interest or direct transfer of the DNA to the skin represents a potential therapeutic approach for the systemic delivery of active molecules. Successful cutaneous gene therapy trials, however, require development of protocols for efficient gene transfer to epidermal stem cells, and information about the host immune response to the recombinant polypeptides produced by the implanted keratinocytes. The availability of spontaneous animal models for genodermatoses will validate the gene therapy approach in preclinical trials.     

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444]     

Gene therapy for Parkinson's disease

Gene therapy is a potentially powerful approach to the treatment of neurological diseases. The discovery of neurotrophic factors inhibiting neurodegenerative processes and neurotransmitter-synthesizing enzymes provides the basis for current gene therapy strategies for Parkinson's disease. Genes can be transferred by viral or nonviral vectors. Of the various possible vectors, recombinant retroviruses are the most efficient for genetic modification of cells in vitro that can thereafter be used for transplantation (ex vivo gene therapy approach). Recently, in vivo gene transfer to the brain has been developed using adenovirus vectors. One of the advantages of recombinant adenovirus is that it can transduced both quiescent and actively dividing cells, thereby allowing both direct in vivo gene transfer and ex vivo gene transfer to neural cells. Probably because the brain is partially protected from the immune system, the expression of adenoviral vectors persists for several months with little inflammation. Novel therapeutic tools, such as vectors for gene therapy have to be evaluated in terms of efficacy and safety for future clinical trials. These vectors still need to be improved to allow long-term and possibly regulatable expression of the transgene.     

Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.     

Advances in treatment of neurodegenerative diseases: Perspectives for combination of stem cells with neurotrophic factors

Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, are a group of incurable neurological disorders, characterized by the chronic progressive loss of different neuronal subtypes. However, despite its increasing prevalence among the ever-increasing aging population, little progress has been made in the coincident immense efforts towards development of therapeutic agents. Research interest has recently turned towards stem cells including stem cells-derived exosomes, neurotrophic factors, and their combination as potential therapeutic agents in neurodegenerative diseases. In this review, we summarize the progress in therapeutic strategies based on stem cells combined with neurotrophic factors and mesenchymal stem cells-derived exosomes for neurodegenerative diseases, with an emphasis on the combination therapy.     

Lentiviral vectors for treating and modeling human CNS disorders

Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral-mediated overexpression of mutated human alpha-synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

Lysosomes play a vital role in the maintenance of cellular homeostasis through the recycling of cell constituents, a key metabolic function which is highly dependent on the correct function of the lysosomal hydrolases and membrane proteins, as well as correct membrane lipid stoichiometry and composition. The critical role of lysosomal functionality is evident from the severity of the diseases in which the primary lesion is a genetically defined loss-of-function of lysosomal hydrolases or membrane proteins. This group of diseases, known as lysosomal storage diseases (LSDs), number more than 50 and are associated with severe neurodegeneration, systemic disease, and early death, with only a handful of the diseases having a therapeutic option. Another key homeostatic system is the metabolic stress response or heat shock response (HSR), which is induced in response to a number of physiological and pathological stresses, such as protein misfolding and aggregation, endoplasmic reticulum stress, oxidative stress, nutrient deprivation, elevated temperature, viral infections, and various acute traumas. Importantly, the HSR and its cardinal members of the heat shock protein 70 family has been shown to protect against a number of degenerative diseases, including severe diseases of the nervous system. The cytoprotective actions of the HSR also include processes involving the lysosomal system, such as cell death, autophagy, and protection against lysosomal membrane permeabilization, and have shown promise in a number of LSDs. This review seeks to describe the emerging understanding of the interplay between these two essential metabolic systems, the lysosomes and the HSR, with a particular focus on their potential as a therapeutic target for LSDs.     

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.     

直接转基因技术应用于神经系统基因治疗的研究进展

神经系统疾病的基因治疗是目前神经科学中发展比较迅速的一个领域,近年来研究发现,一此来源于单纯疱疹病毒,腺病毒和腺病毒相关病毒等的重组病毒表达载体能够将外源基因直接导入在体或离体培养的神经细胞。