Models to study basic and applied aspects of lysosomal storage disorders

Models to study basic and applied aspects of lysosomal storage disorders Ángel Gaudioso a , Teresa P. Silva b , María Dolores Ledesma a, ⇑ a Centro Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain b Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Portugal article info Article history: Received 29 October 2021 Revised 5 August 2022 Accepted 4 September 2022 Available online 16 September 2022 Keywords: Lysosomes Storage disorders Omics Optogenetics iPSCs Organoids BBB Animal models abstract The lack of available treatments and fatal outcome in most lysosomal storage disorders (LSDs) have spurred research on pathological mechanisms and novel therapies in recent years. In this effort, experimental methodology in cellular and animal models have been developed, with aims to address major challenges in many LSDs such as patient-to-patient variability and brain condition. These techniques and models have advanced knowledge not only of LSDs but also for other lysosomal disorders and have provided fundamental insights into the biological roles of lysosomes. They can also serve to assess the efficacy of classical therapies and modern drug delivery systems. Here, we summarize the techniques and models used in LSD research, which include both established and recently developed in vitro methods, with general utility or specifically addressing lysosomal features. We also review animal models of LSDs together with cutting-edge technology that may reduce the need for animals in the study of these devastating diseases. Ó2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Contents 1. Introduction . . . ........................................................................................................ 2 2. In vitro techniques in LSD research . . . ..................................................................................... 2 2.1. ‘‘Omics” . . . . . . . . . . . . . . ........................................................................................... 2 2.2. Fluorescence-based techniques . . . . . . . . . . . ........................................................................... 5 2.3. Electron microscopy . . . . ........................................................................................... 7 2.4. Optogenetics . . . . . . . . . . ........................................................................................... 7 2.5. Liposome-based techniques . . . . . . . . . . . . . . ........................................................................... 8 3. Cellular models in LSD research . . . . . . ..................................................................................... 8 3.1. Primary cell cultures . . . . ........................................................................................... 8 3.2. Immortalized cell lines . . ........................................................................................... 8 3.3. Induced pluripotent stem cells (iPSCs) . . . . . ........................................................................... 9 3.4. Multilineage cellular models: Organoids . . . . .......................................................................... 10 3.5. BBB models . . . . . . . . . . . .......................................................................................... 10 4. Animal models in LSD research. . . . . . . .................................................................................... 11 4.1. Naturally occurring animal models . . . . . . . . .......................................................................... 11 4.2. Drosophila melanogaster .......................................................................................... 11 4.3. Caenorhabditis elegans . . .......................................................................................... 11 https://doi.org/10.1016/j.addr.2022.114532 0169-409X/Ó2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Abbreviations: AAV9, Adenoassociated Viral Vectors serotype 9; ASM, Acid Sphingomyelinase; ASMko, Acid Sphingomyelinase Knock-out; BBB, Blodd-Brain Barrier; BODIPY, Boron dipyrromethene; CNS, Central Nervous System; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; EM, Electron Microscopy; ER, Endoplasmic Reticulum; GAL4/UAS, GAL4/Upstream Activating Sequence; GFP, Green Fluorescent Protein; hiPSCs, human induced Pluripotent Stem Cells; iPSCs, induced Pluripotent Stem Cells; LAMP1/2, Lysosomal Associated Membrane Protein 1/2; LPC, Lysophosphatidylcholine; LPS, Lysophosphatidylserine; LSD, Lysosomal Storage Disorder; mTORC1, mammalian Target Of Rapamicine Complex 1; NBD, Nitrobenzoxadiazole; OMIM, Online Mendelian Inheritance in Man; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PC-O, Plasmenylcholine; PE-O, Plasmenylethanolamine. ⇑ Corresponding author at: Centro Biologia Molecular Severo Ochoa, Nicolas Cabrera 1, 28049 Madrid, Spain. E-mail address: [email protected] (M. Dolores Ledesma). Advanced Drug Delivery Reviews 190 (2022) 114532 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/adr 4.4. Zebrafish . . . . . . . . . . . . . .......................................................................................... 12 4.5. Murine models . . . . . . . . .......................................................................................... 13 4.6. Non-human primates . . . .......................................................................................... 13 5. Conclusion . . . . ....................................................................................................... 13 Declaration of Competing Interest . . . . .................................................................................... 13 Acknowledgements . . . . . . ............................................................................................. 14 Funding . . . . . . . . ....................................................................................................... 14 References . . . . ....................................................................................................... 14 1. Introduction Due to their key role in the breakdown of cellular components [1], and for many years since their discovery in 1950 [2], lysosomes were considered merely ‘‘cellular trash cans”. Nowadays, a plethora of functions beyond the degradation of intraand extracellular molecules and organelles [3] have been assigned to lysosomes, including nutrient sensing [4], the quality control system involved in the ageing process [5] and participation in innate and adaptive immunity [6,7] (Fig. 1). Aside from their involvement in multiple cellular processes, lysosomes make physical connections with several intracellular organelles such as mitochondria [8], the endoplasmic reticulum [9] or plasma membrane [10] by membrane tethering and fusion. These contact sites are crucial for the correct function of not only lysosomes, but also the subcellular organelles that fuse with them [11,12]. Proper lysosomal activity is vital for adequate cell, tissue and organ functions, and lysosomal impairment plays a relevant pathological role in many different diseases including cancer [13] and neurodegenerative [14–16] or autoimmune diseases [17]. Among the lysosome-related diseases are the lysosomal storage disorders (LSDs), whose aetiology is directly linked to lysosomal dysfunction. Most LSDs are caused by mutations in genes that encode lysosomal enzymes, resulting in more than 70 different rare diseases characterised by the lysosomal accumulation of molecules, in many cases the very substrates of the defective enzymes [18]. One way to classify LSDs is according to the type of molecule that accumulates in the lysosomes. This classification scheme (Table 1), which will be used to organize this review, includes three major groups: sphingolipidoses, mucopolysaccharidoses and glycoproteinoses/oligosaccharidoses [53] (Fig. 1). Sphingolipidoses are caused by defects in the lysosomal degradation of sphingolipids, and the accumulation of lipid in these diseases causes membrane defects that can affect cell survival, especially of neurons. As a result, neurodegeneration along with visceral complications are characteristic of many sphingolipidoses [54]. Mucopolysaccharidoses are the consequence of defects in the degradation of glycosaminoglycans (complex amino-sugar polymers). These diseases progress with both peripheral (i.e. upper airway obstruction or cardiac valve and muscle disease) and neuropathic (i.e. hyperactivity, impaired development and loss of cognitive function) features [55]. The third group, glycoproteinoses/oligosaccharidoses result from alterations in catabolism of oligosaccharide chains from glycolipids and glycoproteins, leading to the accumulation of these molecules. Their clinical manifestations are similar to those already mentioned for the mucopolysaccharidoses [56]. Besides the LSDs directly caused by the impaired function of lysosomal metabolic enzymes, some LSDs are the consequence of alterations in proteins that control trafficking of molecules through the endolysosomal compartment [57] or in lysosomal membrane proteins. This group of disorders are also characterized by storage inside lysosomes. Representative examples are Niemann-Pick type C (OMIM 257220; 607625), which results from mutations in the cholesterol transport proteins NPC1 or NPC2 [58,59] or Danon disease (OMIM 300257), which is caused by mutations in the gene encoding the integral lysosomal membrane protein LAMP2 [60]. Identification of the genetic cause in many LSDs has not been accompanied by a deep knowledge of the pathological mechanisms, much less by efficient treatments. Among the important caveats of current LSD therapies is the delivery of therapeutics for these diseases. Drug delivery systems may target and traffic to lysosomes after entering cells. However, accumulation of therapeutics in these organelles prior to degradation could cause deleterious effects. In addition, drug delivery systems that aim to disturb the endolysosomal compartment, so that the contained therapeutics can escape into the cytosol and reach other intracellular targets, may also affect lysosomal function. Particularly challenging is the delivery of therapeutics into the brain, which is necessary to treat the numerous neurological LSDs. Established and newly developed techniques and suitable cellular and animal models are required not only to understand pathological mechanisms in LSDs but also to assess lysosomal status and blood brain barrier penetrance and to evaluate therapeutic strategies and drug delivery systems in these diseases. This review focuses on the current state of such techniques and models, considers advantages and drawbacks (Fig. 2) giving examples of their use in different types of LSDs in the past, and discusses in some instances the potential to advance therapeutic drug delivery and development in the future. 2. In vitro techniques in LSD research Numerous in vitro approaches exist that can be applied to study lysosomal structure and function, as well as to diagnose and determine pathological alterations and therapeutic efficacy for LSDs. In this section we summarise the established approaches that are employed in the LSD context and techniques that have more recently been developed to specifically address lysosomes and their alterations. 2.1. ‘‘Omics” ‘‘Omics” is a group of techniques used to collectively characterize and quantify pools of biological molecules, and they can be applied to a great variety of samples including cultured cells, animal models or patient-derived samples. Depending on the type of biological molecules that are characterized, ‘‘omics” can be divided in proteomics, lipidomics, genomics, metabolomics, etc. ‘‘Omics” tools are very useful in human clinical research because they allow a general characterization of disease-related alterations in different tissues and samples. In the case of genetic diseases like LSDs, genomic analysis can serve to detect key mutations. Additionally, proteomic and/or lipidomic analysis, which detect alterations in the levels of proteins or lipids, respectively, can give a comprehensive view of substrates that may accumulate in LSDs. Other important applications of ‘‘omics” in LSD research are those Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 2 Fig. 1. A. Schematic of known lysosomal functions. B. LSD classification and different types of lysosomal anomalies found in the LSD context indicating the techniques that may help to analyse them. EM: Electron microscopy. Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 3 aimed at finding biomarkers for diagnosis and assessment of treatment efficacy. Given the versatility and insight that ‘‘omics” can provide, there are numerous studies using these techniques in the LSD context. In the sphingolipidoses, proteomic analysis of urine, blood and tissue samples from patients contributed to the finding of new biomarkers in Fabry disease (OMIM 301500) [61]. Proteomic profiling of lysosomes in Niemann-Pick type C (OMIM 257220; 607625), in which sphingolipids also accumulate, identified mTORC1 signalling as a targetable pathway for treatment [62]. Lipidomic analysis enabling the simultaneous quantification of lysosphingolipids allowed the screening of Fabry (OMIM 301500), Gaucher (OMIM 230800), infantile Krabbe (OMIM 245200) and Niemann-Pick diseases with high sensitivity and specificity [63]. This analysis permitted the discovery of N-acyl-O-phosphocholineserine, a class of lipid that had not been previously detected in biological samples, as a biomarker in plasma from Niemann-Pick type C (OMIM 257220; 607625) patients [64]. Lipidomic profiling of plasma and urine from Gaucher disease (OMIM 230800) patients was used to identify lipid species, which levels were significantly altered and could be modified by enzyme replacement therapy indicating that these species were directly or indirectly affected by the therapy [65]. Levels of plasma glucosylsphingosine are being used as reliable marker to follow therapeutic response in Gaucher (OMIM 230800) patients [66]. An example of lipidomic analysis unveiling differences in the brain phospholipid content in a mouse model for the acid sphingomyelinase deficiency is shown in Fig. 3. In mucopolysaccharidoses, brain transcriptomic analysis assessed the effects of central nervous system (CNS) gene therapy in a mouse model for Hunter syndrome (OMIM 309900). Almost 80 % of the genes differentially expressed, of which many associated to inflammation and innate immunity, were corrected at a significant extent in their transcript levels by administration of adeno-associated virus vectors encoding iduronate-2-sulfatase Table 1 List of representative LSDs, classified according to the nature of the storage molecule, indicating the gene affected, the encoded protein, the OMIM (Online Mendelian Inheritance in Man) identifier and the primary accumulating substrate in each of them. *Pompe disease is included in this group due to the accumulation of polysaccharides. Disease Gene affected OMIM Encoded protein Primary accumulating molecule Ref Sphingolipidoses GM1 gangliosidosis GLB1 230,500 b-galactosidase GM1 ganglioside [19] Sandhoff disease HEXB 268,800 Hexosaminidase A/B GM2 ganglioside [20] Tay-Sachs disease HEXA 272,800 Hexosaminidase A GM2 ganglioside [21] Fabry disease GLA 301,500 a -galactosidase A Globotriaosylceramide [22] Metachromatic leukodystrophy ARSA; PSAP 250,100 Arylsulfatase A Sulfatides [23] Krabbe disease GALC 245,200 b-galactocerebrosidase Psychosine [24] Niemann-Pick A/B disease SMPD1 257200; 607,616 Acid sphingomyelinase Sphingomyelin [25] Farber disease ASAH1 228,000 Acid ceramidase Ceramide [26] Gaucher disease GBA 230,800 Glucocerebrosidase Glucosylceramide [27] Mucopolysaccharidoses Hunter syndrome IDS 309,900 Iduronate 2-sulfatase Heparan and dermatan sulfate [28] Hurler-Scheie syndrome IDUA 607,015 a -l-iduronidase Heparan and dermatan sulfate [29] Sanfilippo syndrome A SGSH 252,900 Sulfamidase Heparan sulfate [30] Sanfilippo syndrome C HGSNAT 252,930 Acetyl CoA a -glucosaminide acetyltransferase Heparan sulfate [30] Sanfilippo syndrome B NAGLU 252,920 a -N-acetylglucosaminidase Heparan sulfate [30] Sly disease GUSB 253,220 b-glucuronidase Chondroitin, heparan and dermatan sulfate [31] Sanfilippo syndrome D GNS 252,940 N-acetylglucosamine-6-sulfatase Heparan sulfate [30] Morquio A syndrome GALNS 253,000 N-acetylglucosamine-6-sulfate sulfatase Keratan and chondroitin-6-sulfate [32] Morquio B syndrome GLB1 253,010 b-galactosidase Keratan and chondroitin-6-sulfate [33] Maroteaux-Lamy syndrome ARSB 253,200 Arylsulfatase B Dermatan and chondroitin-4-sulfate [34] Natowicz syndrome HYAL1 601,492 Hyaluronidase 1 Hyaluronan [35] Glycoproteinoses/Oligosaccharidoses Sialidosis type I NEU1 256,550 Neuraminidase Sialyloligosaccharides [36] Sialidosis type II NEU1 256,550 Neuraminidase Sialyloligosaccharides [37] Galactosialidosis CTSA 256,540 Cathepsin A Sialyloligosaccharides [38] Schindler disease NAGA 609,241 a -N-acetylgalactosaminidase Galactose oligosaccharides, galactomannans and galactolipids [39] a -mannosidosis MAN2B1 248,500 a - D -mannosidase Mannose-containing oligosaccharides [40] b-mannosidosis MANBA 248,510 b-mannosidase Diand trisaccharides with b-linked mannose at the non-reducing end [41] Fucosidosis FUCA1 230,000 a - L -fucosidase Fucosylated glycoproteins [42] Aspartylglycosaminuria AGA 208,400 Glycosylasparaginase Glycoasparagines [43] Pompe disease* GAA 232,300 Glucosidase Alpha, Acid Glycogen [44] Non-enzymatic LSDs Danon disease LAMP2 300,257 Lysosome Associated Membrane Protein 2 Glycogen [45] Niemann Pick type C NPC1 NPC2 257220; 607,625 Niemann Pick type C1 Niemann Pick type C2 Cholesterol [46] Salla disease SLC17A5 604,369 Sialin Sialic acid [47] Cystinosis CTNS 219,800 Cystinosin Free cystine [48] Mucolipidosis IV MCOLN1 252,650 Mucolipin-1 Phospholipids, gangliosides and mucopolysaccharides [49] Batten disease CLN3 204,200 CLN3 Lipofuscin-like material [50] Ceroid lipofuscinosis type 7 MFSD8 610,951 Major facilitator superfamily domaincontaining protein 8 Lipofuscin-like material [51] Ceroid lipofuscinosis type 12 ATP13A2 606,693 Polyamine-transporting ATPase 13A2 Lipofuscin-like material [52] Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 4 [67]. Whole exome sequencing screened missense changes in the SGSH gene in a group of adult onset Sanfilippo syndrome patients that presented very mild symptoms raising awareness of this unusual phenotype [68]. In glycoproteinoses, a based-electrospray mass spectrometry technique for the detection of carbohydrates was used to screen a complex mixture of O-glycosylated sialylated amino acids from urine of a Schindler disease (OMIM 609241) patient [69] although this method has not been widely adopted for diagnosis. Transcriptomic analysis of FUCA1 knock-down keratinocytes unveiled the altered expression of genes mainly related to differentiation and immune response in fucosidosis (OMIM 230000) [70]. 2.2. Fluorescence-based techniques Fluorescence-based techniques have greatly facilitated the detection of lysosomal damage and storage by microscopy or spectrophotometry. Lysosomes can be visualized by immunofluorescence techniques using antibodies against lysosomal membrane proteins such as LAMP1 (Fig. 4A). Cell-permeable fluorescent probes are available that target lysosomes. For example, the lysotracker red dye stains acidic compartments in live cells [71], and has been used to monitor lysosomal permeabilization in LSD cellular models [72,73] (Fig. 4B). SiR-lysosome is a fluorogenic and highly specific probe for lysosomes based on pepstatin A, a natural Fig. 2. Illustration of cellular and animal models used in LSD research indicating their main advantages (in green) and drawbacks (in red). Common advantage of the cellular models is the suitability to screen drugs and drug delivery systems and characterize pathological molecular mechanisms. Common disadvantages are the isolation from the physiological environment and, except for primary cultures, the genotypic/phenotypic variations that may arise during the culturing process. Common advantage of animal models is the possibility of studying pathology and therapy in the context of the whole organism. However, species-specific differences in physiology, metabolism and genetics must be taken into account when extrapolating results to the human condition. hiPSCs: Human-induced pluripotent stem cells; BBB: Blood Brain Barrier. Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 5 cathepsin D-binding product [74]. It stains lysosomes in live cells without the need for genetic manipulation or overexpression, and has been used to monitor lysosome motility by singleparticle tracking [75]. Fluorescence-based techniques are also available to monitor lysosomal function. Ratiometric assessment of lysosome pH using exogenous or genetically encoded probes can be used not only to determine lysosomal pathology in LSD models but also to assess the safety of drug delivery systems targeting the lysosome [76]. Newly developed exogenous probes to quantify lysosome pH, such as CQ-Lyso, utilize a single excitation wavelength minimizing background noise and autofluorescence interference. New genetically encoded pH lysosome biosensors allow measurement of lysosomal pH over extended periods of time although it is necessary to assess whether their overexpression has any effect on lysosomal size, positioning or activity [76]. There are different ways to associate fluorescence with the accumulation of specific lipids in the LSD context, including fluorescent analogues of native lipids (e.g. nitrobenzoxadiazole [NBD] or boron-dipyrromethene [BODIPY-lipids]) [77,78]; autofluorescent molecules such as the cholesterol-binding antibiotic filipin [79], or peptide sequences in toxins that mediate specific lipid binding. Examples of the latter are the earthworm toxin lysenin, which binds sphingomyelin [80] (Fig. 5A,B); the D4 domain of perfringolysin O, which binds cholesterol [81]; and the B subunit of cholera toxin, which binds the ganglioside GM1 [82]. Detection of these toxin-derived peptides by specific antibodies, transfection of their cDNAs fused with fluorescent proteins or by adding to them fluorescent tags, has helped characterize lysosomal storage in Niemann-Pick diseases [83,84]. They also allowed the discovery of lipid storage beyond lysosomes, which affects other cellular compartments with key relevance to pathology such as that of sphingomyelin at the plasma membrane in Niemann-Pick type A (OMIM 257200) neurons [80] (Fig. 5A). The aforementioned probes have been used to determine the efficacy of potential therapeutic strategies. Reduction in filipin staining confirmed the usefulness of the cholesterol-extracting drug methyl-b-cyclodextrin [85] or the pharmacological activation of the cholesterol degrading enzyme CYP46 [84] in cellular and animal models for Niemann-Pick type C (OMIM 257220; 607625). Fluorescent-labelled cholera toxin subunit B unveiled Fig. 3. Lipidomic analysis of the altered phospholipid composition in the cerebellum of a mouse model for Niemann Pick type A (ASMko). Phospholipid composition of the cerebral cortex was analyzed by mass spectrometry in different mice (4 wt and 4 ASMko) at 4 months of age. Differences in the levels of the indicated phospholipid species (PC; PE; PS; LPC; LPE; PC-O; PE-O) are represented for each individual mouse as a heatmap expressing the differences between the groups after normalization by using MetaboAnalyst 5.0 software. Fig. 4. Study of lysosomal alterations by microscopy. A: Immunofluorescence against the lysosomal membrane protein LAMP1 in cultured primary neurons from wt mice and from ASMko mice that mimic Niemann Pick disease type A. The fluorescence microscopy image illustrates the increase in lysosomal size due to lipid accumulation in the ASMko neurons compared to wt. White lines delineate the neurons. Scale bar = 10 l m. B: Fluorescence microscopy image of the lysotracker red dye in cultured primary neurons from wt mice treated with vehicle or with 40 l M sphingomyelin to increase the levels of this lipid as in the ASMko neurons. Lysotracker, which stains acidic compartments, shows a punctate pattern corresponding to intact lysosomes in the vehicle treated cells while having a diffuse cytosolic display in the SM treated cells indicating lysosomal permeabilization. White lines delineate the neurons. Dapi in blue indicates nuclei. Scale bar = 10 l m. C: Electron microscopy micrographs of microglia in WT and ASMko mice. The images allow visualization of expanded lysosomes and vacuoles in the cytosol of the ASMko microglia compared to the wt. Scale bar = 2 l m. Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 6 the effects of cyclodextrin on GM1-ganglioside levels in cellular models of gangliosidosis (OMIM 230500) [86]. BODIPY-labeled C12 sphingomyelin was used as a substrate to detect acid sphingomyelinase (ASM) activity in cerebrospinal fluid and plasma after adeno-associated viral vector-based gene therapy in the ASM knock-out mouse line that models Niemann-Pick type A (OMIM 257200) [87]. Fluorescence-based methods have been key in the clinical diagnosis of LSDs, which heavily relies on either detection of the accumulation of molecules or identification of defective activity of enzymes in patient cells and fluids. Staining of the autofluorescent antibiotic Filipin in patient fibroblasts has been traditionally used for the diagnosis of Niemann-Pick type C (OMIM 257220; 607625) [88]. The fluorescent-labelled ganglioside sulforhodamine-GM1 has been used to determine the activity of hexosaminidase A in skin fibroblasts and white blood cells from patients of GM2-gangliosidosis (OMIM 272800), allowing researchers to distinguish between the various subtypes of this LSD [89]. Keratin sulfate-dependent fluorescence has been used to diagnose Morquio A syndrome (OMIM 253000) from dried blood samples [90]. A simple method determined the enzymatic activity of a -L-fucosidase by measuring the fluorescence quenching of CdTe semiconductor quantum dots in serum samples from fucosidosis (OMIM 230000) patients [91]. 2.3. Electron microscopy Although less amenable than fluorescence-based microscopy methods, electron microscopy (EM) has also been widely used to analyse the ultrastructural changes related to the deposit of nondegraded molecules within the lysosomes and cytosol of cells from patients and LSD animal models (Fig. 4C). EM analysis of cells and tissues crucially contributed to the identification of autophagy alterations as a common pathological feature in many LSDs [92,93]. Single-particle cryo-EM has yielded protein structures with increasing levels of detail in recent years, and it can be applied to explore the dynamic conformational changes of lysosomal channels and proteins. Although EM does not have the scaling and output to be adopted for high throughput screening it has risen as a complementary method to traditional structural techniques and does show promise in structure-based drug discovery [94]. As EM has been used in the study of almost all LSDs, here we only wish to highlight a few relevant examples, mainly in human tissue. In sphingolipidoses, a recent comprehensive ultrastructural examination of the autopsy tissue from a Niemann-Pick type A (OMIM 257200) patient reported significant accumulation of sphingomyelin even in organs that did not show overt clinical manifestations [95]. Inclusions within the cytoplasm of podocytes were observed in a patient carrying unidentified gene variation associated with Fabry disease (OMIM 301500) [96]. EM carried out on skin biopsy samples from Farber disease (OMIM 228000) patients harbouring a new mutation revealed the presence of stacks of membranes in enlarged lysosomes also known as zebra bodies [97]. In mucopolysaccharidoses, membrane-bound vacuoles and deposits of sulfated acidic glycosaminoglycans were detected by EM in biopsied muscles from a Hunter syndrome (OMIM 309900) patient [98]. Keratocytes containing vacuoles and electron-dense lysosomes full of proteoglycans were described in the cornea from a patient of Maroteaux-Lamy syndrome (OMIM 253200) [99]. Single-particle cryo-EM helped to resolve the structural differences between distinct conformations of the TRPML1 channel, and may contribute to understand the potential mechanism of different mutations in mucolipidosis type IV (OMIM 252650) [100]. In glycoproteinoses, EM techniques allowed the description of an accumulation of terminal sialic acid at the non-reducing end of the sugar chain in the brain tissue of a sialidosis patient (OMIM 256550) [101]. Distended lysosomes within endothelial cells, fibroblasts and histiocytes were found in the skin of a fucosidosis patient (OMIM 230000) [102]. 2.4. Optogenetics Optogenetic methods allow precise manipulation of many cellular activities with light [103]. Through the use of illumination and the expression of photoproteins, protein–protein interactions and biochemical pathways can be controlled. Among the advantages of optogenetic methods are the cell type specificity, the high speed and accuracy and less damage to tissue. Among the main challenges for optogenetic implementation in humans are the expression of adequate amounts of photoproteins and the development of low-heat light sources. Different optogenetic strategies have been used in cellular and animal models to monitor and modulate relevant features of lysosome physiology including their pH or motility. As an example, light activation of lyso-pHoenix, a fusion protein made up of the light-activated proton pump Arch3, the pH-sensitive GFP variant pHluorin and the lysosomal-targeting CD63 protein, caused a rapid drop in lysosomal pH [104]. This probe enables the external control of lysosomal pH and can be applied to directly assess the acid-dependent activity of lysosomal proteases and the role of acidification in LSD pathology. Optogenetic strategies have also been used to control the transport and distribution of lysosomes. Upon light-induced recruitment of molecular motors such as kinesin or dynein, repositioning of these organelles in cells has been achieved [105]. Optogenetic modules Fig. 5. Primary cell cultures to study lipid alterations in LSDs. A: Surface staining in non-permeabilized primary cultured neurons from WT mice and ASMko mice, which mimic Niemann Pick disease Type A, using an antibody against the toxinderived peptide Lysenin that specifically binds sphingomyelin. This kind of experiment showed that in ASMko neurons sphingomyelin accumulation not only occurs in the lysosomes but also at the plasma membrane. Scale bar = 5 l m. B: Lysenin staining in permeabilized cultured fibroblast obtained from a control subject and a Niemann Pick disease type A patient evidencing the intracellular accumulation of sphingomyelin in the patient cell. White circles indicate the nuclei. Scale bar = 10 l m. Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 7 based on light-sensitive dimerizers have been used to translocate mTORC1 to lysosomes upon photoactivation [106]. Phototoxic proteins have been targeted to the cytoplasmic surface of lysosomes by fusion with the small GTPase Rab7. This strategy has been used to induce lysosomal membrane permeabilization [107] or oxidative stress [108] upon illumination. 2.5. Liposome-based techniques Liposome-based techniques have contributed to both characterization and treatment of pathology in LSD models. Liposomes are spherical particles made up of different types of lipids that organise to form one or more lipid bilayers. They can be synthesized by different means [109] including the mechanical methods of thin film hydration [110] or solvent dispersion [111]. The broad application of liposomes mainly derives from their capacity to encapsulate different types of compounds or genetic vectors [112]. Liposomes can deliver their cargo to almost all organs and tissues. This includes the brain when strategies like coating or conjugation with antibodies are used to help liposomes cross the blood–brain barrier (BBB) [113]. Several key features must be determined in vitro before applying liposomes in clinical research settings, including solubility, stability, cargo uptake, ability to cross endothelial barriers and BBB structures, and effective delivery of cargo into the desired cell/organelle. We refer to the articles in this issue by Drs. Ventosa, Ceccini and Muro for excellent reviews on the use of liposomes and other nanoparticles in LSD therapy. In addition to therapeutics, liposome-based techniques have been used as tools to study LSD pathological mechanisms and to create LSD models. In sphingolipidoses, a detergent-free liposomal assay uncovered the regulatory roles of distinct lipid classes on the hydrolysis of membrane-bound glucosylceramide by the lysosomal b-glucocerebrosidase in Gaucher disease (OMIM 230800) models [114]. Reconstitution of GM2 catabolism at liposomal surfaces was used as a model to study the effect of the accumulation of different membrane lipids on ganglioside metabolism and gangliosidosis [115]. In glycoproteinoses, liposomes loaded with glycolipids were used to demonstrate the deficient degradation of these lipids by skin fibroblasts from patients of Schindler disease (OMIM 609241) [116]. 3. Cellular models in LSD research Cellular models have been commonly used in LSD research. From traditional monocultured cells in isolation to cutting-edge technologies leading to the generation of multi-lineage cell platforms, these techniques have facilitated our understanding of pathological molecular mechanisms and driven the discovery of drugand cell-based therapies. 3.1. Primary cell cultures Primary cell culture is defined as the ex vivo culture of cells freshly obtained from a tissue. The main advantages of primary cells are their faithful transcriptomic and proteomic profile and closer proximity to physiological function and response. Some drawbacks are their requirements for specific substrates and nutrients and the acquisition of a senescent phenotype, which leads to an irreversible cell cycle arrest that limits the amount of material that can be obtained. This prevents the use of primary cultures for large-scale disease modelling and drug discovery projects. Primary cultures of fibroblasts from patient-derived skin biopsies have been broadly used in LSDs, not only to understand disease mechanisms but also for diagnostic purposes (Fig. 4B). In mucopolysaccharidoses, transcriptomic studies on fibroblast cultures derived from patients suffering from all types of these diseases identified dysregulation of apoptosis-related genes [117].In glycoproteinoses, the use of primary cultured fibroblasts from patients suffering from sialidosis (OMIM 256550) and galactosialidosis (OMIM 256540) demonstrated impaired elastogenesis that could be reverted by transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase [118]. Filipin staining to detect cholesterol accumulation in primary fibroblasts from Niemann-Pick type C (OMIM 257220; 607625) patients has been a key diagnostic tool for decades [119]. The use of primary neuronal cultures has been particularly informative for the neurological phenotypes in LSDs. Both monocultures and co-cultures of primary brain cells obtained from mouse models have been used. In sphingolipidoses, primary cultured neurons from ASM knock-out mice, which mimic Niemann-Pick type A (OMIM 257200) (Fig. 4A), unveiled alterations in autophagy [72], calcium homeostasis [80] and synaptic features [120,121]. In mucopolysaccharidoses, co-cultures of primary brain cells from mice lacking sulfamidase (which mimic Sanfilippo syndrome (OMIM 252900)) revealed the highest accumulation of heparan sulfate in astrocytes [122]. 3.2. Immortalized cell lines Immortalized cell lines are generated by naturally occurring or induced mutations that avoid senescence and allow cells to keep dividing. They can be used in research instead of primary cultures, providing pure populations of cells and more reproducible results. Among other advantages, they are cost effective, deliver an almost unlimited supply of material and bypass the ethical concerns associated with the use of animal and human tissue. However, due to the genetic manipulation that is required to generate them, immortalized cell lines may show altered phenotype when compared to primary cultures. Serial passage can also cause genotypic and phenotypic variations and contamination with mycoplasma is common [123]. Despite these drawbacks, immortalized cell lines have been instrumental in LSD research. They may express specific lysosomal enzymes to be used as therapeutic tools. Moreover, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, firstly discovered as a defence system in bacteria and archaea [124], facilitates genome editing and is a powerful tool [125] that is increasingly being used to generate LSD disease modelling cell lines. Of particular relevance in the LSD context are immortalized cells generated after transformation of patient-derived primary cells by, for example, hTERT or SV40 large T antigen technology [126]. Comparisons among single-patient derived cell lines are useful to understand the variable phenotype of LSD patients, which often depends on the specific mutations they carry. In sphingolipidoses, a Chinese hamster ovary cell line overexpressing human GM2A was generated to purify the secreted enzyme and study its uptake by fibroblasts derived from Sandhoff disease (OMIM 268800) patients [127]. A human monocytic THP-1 cell line, in which the GBA1 gene was edited, was used to model Gaucher disease (OMIM 230800) [128]. GLA gene knock-out HEK-293T cells were generated by the CRISPR/Cas9 methodology to study the cellular pharmacokinetics of recombinant human a - galactosidase A [129]. The therapeutic potential of gene edited neural stem cells overexpressing galactosylceramidase was shown in Krabbe disease (OMIM 245200) [130]. An NPC1 knock-out HeLa cell line was generated by CRISPR/Cas9 methodology to study Niemann-Pick type C (OMIM 257220) [131]. In mucopolysaccharidoses, cathepsin B leakage from lysosomes to the cytoplasm was reported in a Hunter syndrome (OMIM 309900)-derived neuronal cell line [132]. NAGLU-deficient and Á. Gaudioso, T.P. Silva and Marí. Dolores Ledesma Advanced Drug Delivery Reviews 190 (2022) 114532 8 HGSNAT-mutated cell lines modelling Sanfilippo syndrome types B (OMIM 252920) and C (OMIM 252930), respectively, were generated by CRISPR/Cas9 technology [133,134]. In glycoproteinoses, RAG and LM/TK cell lines contributed to the chromosomal assignment of two genes associated with neuraminidase-deficiency disorders [135]. 3.3. Induced pluripotent stem cells (iPSCs) iPSCs are generated by reprogramming of somatic cells through the manipulation of specific transcription factors and exhibit the morphology, growth properties and marker genes of embryonic stem cells [136,137]. iPSCs can be obtained from different research animals, and even from human biopsies. Human iPSCs (hiPSCs) harbour natural-disease causing mutations and allow study of the role of genetic background in disease progression and severity. Importantly, they can be differentiated to produce cellular lineages that cannot be obtained directly from patients, such as neurons or neural stem cells. Therefore, they are instrumental to study the neurological phenotypes that characterize many LSDs (Table 2). Given their inherent patient specificity, hiPSC-derived cell types may be key to develop personalized drug treatments [163]. Efforts are currently underway to reduce the variability of directed hiPSC differentiation, which can lead to populations of multiple cell types that arise spontaneously [164]. Use of human iPSCs in LSD research has been extensively reviewed in [165]. Here, we provide only some examples that highlight their utility. In sphingolipidoses, oxidative stress and presynaptic dysfunction were described in neurons created from iPSCs obtained from skin-derived fibroblast of patients with Tay-Sachs disease (OMIM 272800) [142]. Patient-derived iPSCs and geneediting technology were used to study the cardiac-related molecuTable 2 List of iPSCs and organoids generated for LSD research. hiPSCs: Human-induced pluripotent stem cells. iPSCs: Induced pluripotent stem cells. ND: Not described. Disease iPSCs Organoids Sphingolipidoses GM1 gangliosidosis Neurons differentiated from patient-derived hiPSCs [138] Cerebral organoids from iPSCs [139] Sandhoff disease iPSCs derived from mouse model neural stem cells [140] Cerebral organoids from patient-derived hiPSCs [141] Tay-Sachs disease hiPSCs from patient-derived fibroblasts [142] Cerebral organoids from patient-derived hiPSCs [141] Fabry disease hiPSCs from patient-derived fibroblasts [143] Metachromatic leukodystrophy hiPSCs line from patient-derived fibroblasts [144] Artificially iPSCs-derived whole-brain organoid [145] Krabbe disease hiPSCs line from patient-derived fibroblasts [146] ND Niemann-Pick A/B disease hiPSCs line from type A patientderived dermal fibroblasts [147] hiPSCs line from type B patientderived dermal fibroblasts [148] ND Farber disease hiPSCs line from patient-derived fibroblasts [149] ND Gaucher disease hiPSCs line from patient-derived fibroblasts [150] ND Mucopolysaccharidoses Hunter syndrome hiPSCs from patient-derived dermal fibroblasts [151] ND Hurler-Scheie syndrome hiPSCs from patient-derived fibroblasts [152] ND Sanfilippo syndrome C HGSNAT-mutated cell lines from healthy hIPSCs [134] ND Sanfilippo syndrome B hiPSCs from patient-derived peripheral blood mononuclear cells [153] ND Sly disease hiPSCs from patient-derived fibroblasts [154] ND Morquio A syndrome hiPSCs from patient-derived dermal fibroblasts [155] ND Glycoproteinoses/Oligosaccharidoses Sialidosis type I IPSCs-derived neural cells generated from patient-derived fibroblasts [156] ND Sialidosis type II IPSCs-derived neural cells generated from patient-derived fibroblasts [156] ND Non-enzymatic LSDs Niemann Pick type C Neurons differentiated from i 3 Neuron iPSCs [157] hiPSCs from patient-derived fibroblasts [158] Cerebral organoids from patient-derived hiPSCs [159] Danon disease hiPSCs from patient-derived peripheral blood mononuclear cells [160] ND Batten disease Gene correction studies in hiPSCS from patient-derived dermal fibroblasts [161] Cerebral organoids from patient-derived hiPSCs [162] Á. 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