key: cord-268326-sbz3uk5h
authors: Bonam, Srinivasa Reddy; Wang, Fengjuan; Muller, Sylviane
title: Lysosomes as a therapeutic target
date: 2019-09-02
journal: Nat Rev Drug Discov
DOI: 10.1038/s41573-019-0036-1
sha: 
doc_id: 268326
cord_uid: sbz3uk5h

Lysosomes are membrane-bound organelles with roles in processes involved in degrading and recycling cellular waste, cellular signalling and energy metabolism. Defects in genes encoding lysosomal proteins cause lysosomal storage disorders, in which enzyme replacement therapy has proved successful. Growing evidence also implicates roles for lysosomal dysfunction in more common diseases including inflammatory and autoimmune disorders, neurodegenerative diseases, cancer and metabolic disorders. With a focus on lysosomal dysfunction in autoimmune disorders and neurodegenerative diseases — including lupus, rheumatoid arthritis, multiple sclerosis, Alzheimer disease and Parkinson disease — this Review critically analyses progress and opportunities for therapeutically targeting lysosomal proteins and processes, particularly with small molecules and peptide drugs.

Discovered in the 1950s by Christian de Duve, lyso somes are membrane bound vesicles containing numerous hydrolytic enzymes that can break down biological polymers such as proteins, lipids, nucleic acids and polysaccharides 1,2 . Lysosomes have long been known to have a key role in the degradation and recycling of extracellular material via endocytosis and phagocytosis, and intracellular material via autophagy (reviewed elsewhere 2-5 ) (Fig. 1 ). The products of lyso somal degradation through these processes can be trafficked to the golgi apparatus for reuse or for release from the cell through lysosomal exocytosis, which is important in immune system processes. In addition, it has become clear more recently that lysosomes have an important role in other cellular processes including nutrient sensing and the control of energy metabolism 3,5-7 (Fig. 1) .

Alterations in lysosomal functions, either in the fusion processes involved in the general pathways mentioned above or related to the function of lyso somal enzymes and non enzymatic proteins, can result in broad detrimental effects, including failure to clear potentially toxic cellular waste, inflammation, apopto sis and dysregulation of cellular signalling 8 . Such defects have been implicated in many diseases, ranging from rare lysosomal storage disorders (LSDs), which are caused by the dysfunction of particular lysosomal proteins, to more common autoimmune and neurodegenerative dis orders 5, 9, 10 . Despite some limitations, impressive results have been achieved in treating several LSDs through enzyme replacement therapy (ERT). In addition, sub stantial efforts have been focused on therapeutically targeting the autophagy processes upstream of lyso somes [11] [12] [13] [14] . However, there has so far been less attention on investigating the potential to directly target lysosomes with small molecules and peptide drugs.

Nevertheless, with recent advances in understand ing of lysosomal function and dysfunction in dis eases, promising novel opportunities for therapeutic intervention through targeting lysosomes specifically are beginning to emerge. This Review will provide a brief overview of lysosomal biogenesis, structure and function, and describe the role of lysosomal dysfunc tion in LSDs as well as other, more common diseases. Specifically, the article will focus on organ specific and non organspecific autoimmune diseases, including lupus, rheumatoid arthritis (RA) and multiple sclerosis (MS), as these have not been extensively reviewed elsewhere, but will also briefly highlight neurodegen erative disorders such as Alzheimer disease (AD) and Parkinson disease (PD), to further illustrate the breadth and nature of the emerging therapeutic opportunities. The current 'toolbox' of pharmacological agents that modulate lysosomal functions and emerging novel tar gets and strategies in this set of indications will be high lighted. It should be noted that therapeutic approaches to treat inflammatory and autoimmune diseases aim to inhibit the deleterious excessive lysosomal activity, whereas lysosomal activation would be the goal in the treatment of neurodegenerative diseases. Although beyond the scope of this review, such approaches may have applications in other diseases in which lysosomes may play a role, including cancer, metabolic diseases and ageing (reviewed elsewhere 15, 16 ).

The formation of mature lysosomes is a complex process, which involves the fusion of late endosomes that contain material taken up at the cell surface with transport ves icles that bud from the trans Golgi network 5, 8, 17 . These vesicles contain nearly 60 different hydrolytic enzymes (grouped into nucleases, proteases, phosphatases, lipases, Endocytosis A vesicle-mediated process by which cells engulf membrane and extracellular materials. Several endocytic pathwaysphagocytosis, pinocytosis and receptor-mediated endocytosis -utilize different mechanisms to internalize material. Clathrinmediated endocytosis is the major endocytic pathway in mammalian cells. Fig. 1 | The central position of lysosomes at the crossroads of major autophagic pathways. a | Functional lysosomes are involved in the degradation (endocytic and autophagic) and regulation of exogenous and endogenous cellular material, including recycling processes. Extracellular material endocytosed by the endosomes and intracellular cargo internalized by the autophagosomes fuse with lysosomes for degradation, which produces energy (ATP production) and source molecules for the macromolecules. Mechanistic target of rapamycin complex 1 (mTORC1) plays a key role in lysosomal nutrient sensing signals (lysosome-to-nucleus axis) to regulate energy metabolism. Factors such as energy levels, type of pH, ion channel regulation and others decide the fate of the catabolic process. During lysosomal exocytosis, the lysosomal content favours plasma membrane (PM) repair, bone resorption, immune response and elimination of pathogenic stores. b | The lysosome is the ultimate cell compartment that digests unwanted protein materials generated by macroautophagy , microautophagy (pathways during which the cytoplasmic material is trapped in the lysosome by a process of membrane invagination) and chaperone-mediated autophagy (CMA). In general, lipid droplets (LDs) are degraded by lipophagy , a subtype of macroautophagy , which is activated by cytosolic lipases. CMA has also been demonstrated to participate in the degradation of LDs in which perilipin (PLIN2/3) proteins are phosphorylated (P) by AMP-activated protein kinase (AMPK) with the help of the HSPA8 chaperone. Mechanistic target of rapamycin complex 2 (mTORC2) and AKT (also known as protein kinase B) are negative regulators of CMA , where they exert their effect on the translocation complex of CMA. In situations of starvation, negative regulators are controlled by pleckstrin homology domain and leucine-rich repeat protein phosphatase (PHLPP). Lysosomal stability effects the transcription factor EB (TFEB) translation to the nucleus in which TFEB binds to the coordinated lysosomal expression and regulation (CLEAR) motifs to regulate the transcription of genes. EF1a, elongation factor 1a; Lys, lysosome; Rac1, Ras-related C3 botulinum toxin substrate 1.

An endocytic process by which certain cells called phagocytes (for example, macrophages) internalize large particles (>0.5 µm) such as bacteria, other microorganisms, foreign particles or aged red blood cells, for example, to form a phagosome.

A vital, finely-regulated and evolutionarily-conserved intracellular pathway that continuously degrades, recycles and clears unnecessary or dysfunctional cellular components. Autophagy is crucial for cell adaptation to the environment and to maintain cell homeostasis, especially under stress conditions.

Cytosolic apparatus, meant for the regulation of proteins (modification, storing and transportation) and some forms of lipids to the other cytosolic compartments via the trans-golgi network or outside the cell.

A process of the secretory pathway in which lysosomes are fused with the plasma membrane and empty their contents outside the cell. This process plays an important role in plasma membrane repair, bone resorption, immune response and elimination of pathogenic stores (mainly in lysosomal storage disorders). (LSDs) . A group of heterogeneous disorders caused by defects in the lysosomal enzymes leading to the accumulation of unmodified or unprocessed components in the lysosomes, which ultimately influence other vital pathways in the cells. LSDs implicate various vital systems of the human body including the skeleton, brain, skin, heart and central nervous system, which are connected with different metabolic pathways. sulfatases and others), which are synthesized in the endo plasmic reticulum and delivered to the transport vesicles via diverse systems, such as mannose6phosphate tags that are recognized by mannose6phosphate receptors (MPRs) at the membrane 8, 18 or glucocerebrosidase (GCase) that is transported to lysosomes by lysosomal integral membrane protein2, an ubiquitously expressed type III transmembrane glycoprotein mainly located in endosomes and lysosomes 19 .

Mature lysosomes have an acidic internal pH, at which the lysosomal hydrolases are active, and a lin ing known as a glycocalyx that protects the internal lysosomal perimeter from the acidic environment of the lumen 5, 8, 20 . This acidic environment is maintained through the activity of a vacuolar type proton adeno sine triphosphatase (v ATPase), which harnesses energy from hydrolysing ATP to drive the translocation of protons through a V 0 membrane domain (reviewed elsewhere 5, 21 ). Other key lysosomal proteins include struc tural proteins such as lysosomeassociated membrane protein 1 (LAMP1); proteins involved in trafficking and fusion, such as soluble N ethylmaleimidesensitive factor attachment protein receptors (SNAREs) and RAB GTPases; transporters such as LAMP2A, which has a key role in chaperone-mediated autophagy (CMA); and ion channels such as the chloride channel ClC7 and the cation channel mucolipin 1, a member of the transient receptor potential (TRP) family that is also known as TRPML1 (ReFS 22, 23 ). Most of the proteins are deliv ered through the clathrin adaptor protein 3alkaline phosphatase (ALP) pathway, but some proteins are translocated through the lysosome associatedprotein transmembrane5, a protein that is preferentially expressed in immune cells 3, 24 .

Although the concept still remains controversial, two lysosome species -conventional or secretoryare often distinguished based on their physical, bio chemical and functional properties. Catabolism is the main function of conventional lysosomes, and several other lysosome related organelles (LROs), such as mel anosomes, the late endosomal major histocompatibility complex class II (MHCII) compartment (MIIC), lytic granules from neutrophils, eosinophils, basophils, mast cells, CD8 + T cells and platelets, complement these functions 8, [25] [26] [27] [28] [29] . Many of the LROs act as professional secretory organelles. LROs share with lysosomes the majority of typical characteristics (acidic environment, lysosomal transmembrane proteins, fusion property to phagosomes and others), in addition to particular properties resulting from their specific cargoes (for example, melanosomes contain melanosome specific transmembrane glycoprotein, and natural killer cells and CD8 + T cells contain perforins and granzymes). The detailed mechanisms of biogenesis and secretion of LROs remain unclear, although it is known that genetic defects in LROs are involved in rare autosomal recessive disorders characterized by reduced pigmentation, such as Chediak-Higashi disease and Hermansky-Pudlak syndrome 30 . Secretory lysosomes contain many more proteins in addition to those contained in conventional lysosomes, and they participate in multiple cell func tions such as plasma membrane repair, tissue and bone regeneration, apoptotic cell death, cholesterol homeostasis, pathogen defence and cell signalling 8 .

Lysosomal biogenesis and function are regulated by the basic helix-loop-helix leucine zipper transcription factor eB (TFEB) and the coordinated lysosomal expres sion and regulation (CLEAR) network 4,31,32 (Fig. 2) . For example, autophagy, a crucial process in immunity and autoimmunity 33 , is transcriptionally regulated by TFEB 31 . Interestingly, lysosomal exocytosis, which is important in many immune functions, also depends on TFEB activation 31, 32 . Moreover, it has been demonstrated that TFEB orchestrates lysosomal Ca 2+ signalling 34 . The fact that multiple lysosomal processes are dependent on TFEB activation strengthens its role as a master regulator in lysosomal functions. Like other transcription factors, TFEB undergoes phosphorylation and dephosphory lation via different cytosolic and lysosomal pathways (Fig. 2) , processes regulated by mechanistic target of rapamycin complex 1 (mTORC1), a master controller of cell growth 35, 36 .

Lysosomes are at the crossroads of various degrada tive pathways, including endocytosis (phagocytosis) and autophagy (Fig. 1 ). Three main forms of autophagy have been described: macroautophagy (the most extensively characterized form), microautophagy and CMA. At the initiation of macroautophagy, a double membrane sequestering compartment termed the phagophore, which contains cytoplasmic material, is formed and matures into a vesicle called the autophagosome. The cargo is degraded into vacuoles issued from the fusion of autophagic vesicles and lysosomes (called autolyso somes), and the resulting short products are released back into the cytosol for reuse or, according to some times contested observations, possibly dispatched into the MIIC for ultimate processing and MHCII molecule loading for presentation to CD4 + T cells 37, 38 . In contrast to macroautophagy, microautophagy is characterized by direct lysosomal engulfment of cytosolic material into lysosomes, via the formation of characteristic invag inations of the lysosomal membrane. The third major form of autophagy is CMA, which involves the recog nition of substrate proteins containing a KFERQ like motif by a HSPA8/HSC70containing complex (Fig. 1b) . In CMA, two proteins have a key role: HSPA8 ensures the selectivity of proteins, which will be degraded via the CMA pathway; and LAMP2A translocates the tar geted cytosolic proteins across the lysosomal mem brane (reviewed elsewhere 7 ). The terminal step of autophagy is called autophagic lysosome reformation, in which tubular proto lysosomes are extruded from autolysosomes (containing lysosomal membrane com ponents) and mature into functional lysosomes 39 . This step is not solely a lysosomal biogenesis process; it also includes a series of elements that are tightly correlated with the regulation of autophagy 40 .

In combination with autophagy, lysosomes are involved in both innate and adaptive immune func tions, including foreign material recognition (bacterial, parasitic and viral), activation of pattern recognition receptors (such as Toll like receptors (TLRs) and nucleo tide oligomerization domain like receptor), antigen processing and presentation, especially in the context Multiple sclerosis (MS) . A demyelinating disease in which the myelin sheaths wrapped around nerve fibres in the central nervous system are progressively destroyed by immune cells and possibly also by autoantibodies.

Parkinson disease (PD) . A neurodegenerative disorder with symptoms including slowness of movement and a loss of fine motor control, owing to the degeneration of dopamineproducing neurons in the substantia nigra.

(CMA). A selective autophagy pathway in which proteins that contain a signal KFeRQ-like sequence are targeted by HSAP8/HSC70 chaperones and translocated into lysosomes via LAMP2A.

Transcription factor EB (TFeB). A protein that plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. it controls lysosomal function via the coordinated lysosomal expression and regulation (CLeAR) gene network (including genes coding for hydrolases, lysosomal membrane proteins and the proton pump v-ATPase complex), and additional lysosome-related processes such as autophagy, endocytosis and exocytosis.

A finely-regulated process during which the cell forms a double-membrane sequestering compartment named the phagophore, which matures into the autophagosome.

A double membrane-bound vesicle, which encloses cellular constituents and fuses with lysosomes to form phagolysosomes where the engulfed material is digested or degraded and either released extracellularly via exocytosis or released intracellularly to undergo further processing. of MHCII molecules, T cell homeostasis, antibody pro duction and induction of various immune signals (co stimulation and cytokine secretion) 41 . Besides being a degradative organelle, the lysosome has recently been recognized as a cellular signalling platform 3, 42 . It plays an important role in nutrient sensing through mTORC1 and other additional protein complexes, or the so called 'lysosome nutrient sensing machinery' . The discovery of a stress induced lysosome tonucleus signalling mech anism through TFEB further supports the key role of lysosomes in cellular signalling 36 .

The lysosome occupies a central position in the main tenance of cellular homeostasis, being involved in the exclusion of infectious agents from penetrating host tis sue and concomitantly promoting immune regulation. Lysosomes must therefore be able to respond quickly, with increased or decreased functions, to various metabolic conditions aimed at protecting cells from death or damage. Lysosomes are very diverse in size and shape. For reasons that are not totally understood -pos sibly according to their position in the cytosol 43 and/or their composition -some lysosomes in a single cell are more prone to act and defend cells. Given the wide range of functions of lysosomes in all metabolic compartments of the cell, any dysregulation of their activity could lead to the impairment of various elements of the cellular metabolic machinery (including the transport and bio genesis of sugar (glycolysis), lipids, proteins and nucleic acids) and of metabolic pathways, phagocytosis, endo cytosis and autophagy. Although the underlying mech anisms are far from being fully deciphered, it has been seen that lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative dis eases. Dysfunctions of lysosomes can affect the proper activity of other organelles such as peroxisomes and After their synthesis in the rough endoplasmic reticulum (RER), the substrates (cargo) that are intended to be degraded through the endo-lysosomal pathway are transported to lysosomes via the trans-Golgi network (TGN). Among the key enzymatic systems that are involved in the lysosomal enzyme transportation of cargos from Golgi to lysosomes, the best studied is the mannose-6-phosphate (M6P) receptor (MPR) system, which binds newly synthesized lysosomal hydrolases in the TGN and delivers them to pre-lysosomal compartments. A few components synthesized in the late Golgi compartment are delivered directly to lysosomes via the 3-alkaline phosphatase (ALP) pathway. Lysosomal components, such as enzymes (lytic enzymes and kinases), membrane-bound proteins/complexes (mechanistic target of rapamycin (mTOR)), transporters and ion channels (vacuolar-type proton adenosine triphosphatase (v-ATPase), TRPML1 and osteopetrosis associated transmembrane protein 1 (Ostm1)) and chaperone-mediated transportation are the best-known targeting sites for lysosomal dysfunction. As depicted in the figure, many pharmacological antagonists and agonists exert activities that potentially correct lysosomal dysfunction and therefore represent potential effective pharmacological tools. CLEAR , coordinated lysosomal expression and regulation; CQ, chloroquine; HCQ, hydroxychloroquine; mTORC1, mTOR complex 1; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; RAPTOR , regulatory-associated protein of mTOR; SER , smooth endoplasmic reticulum; TFEB, transcription factor EB.

www.nature.com/nrd mitochondria, leading to excessive production of reac tive oxygen species with pathological features associated with ageing, cancer, chronic inflammation, neurological diseases, male infertility and infections. Such dysregulation is thus central to LSDs, and also implicated in a wide range of other disorders, includ ing autoimmune and neurological disorders, in which the autophagy-lysosomal network under the control of TFEB has attracted considerable attention.

LSDs are a heterogeneous group of about 50 inherited metabolic disorders, which have an incidence of ~1 in 5,000 live births 44 . These disorders and their treatment have been reviewed extensively elsewhere 45, 46 , and so will only be covered relatively briefly here. The mutations responsible for most LSDs have been largely elucidated (TABLeS 1,2), and many result in the dysfunction of a particular lysosomal hydrolase, leading to the accumu lation of the substrate of that hydrolase. For example, in Gaucher disease, the sphingolipid glucocerebroside accumulates in cells (particularly macrophages) and organs, including the liver and spleen, owing to defi ciency in the enzyme GCase 24, 66 . In certain LSDs, the resultant pathology can be explained by the nature of molecules that accumulate (TABLeS 1,2). Thus, the abun dance of cerebrosides and gangliosides that deposit in the central nervous system (CNS) of patients with sphingo lipid storage disorders, such as type II (acute infantile neuronopathic) Gaucher disease, underlies the severe neurological symptoms of such disorders 67, 68 . In patients with Pompe disease, which is caused by α glucosidase deficiency, the high levels of non degraded glycogen that accumulate in muscles could explain the observed myopathy 69, 70 . However, how the undegraded material accumulates and causes the observed cellular and organ pathology in many other LSDs remains unclear.

The accumulation of such undigested macromol ecules or monomers in LSDs instigates the formation of secondary products, which ultimately escape from the endosomal-autophagic-lysosomal pathways 9,71 and lead to multiple consequences that affect most organs, including the brain, liver, spleen, heart, eyes, muscles and bone (TABLe 2) . Most, if not all, organelles are altered in LSDs, including endosomes, autophagosomes and lysosomes, and their functions in lysosomal formation/ reformation and fusion of endosomes or autophago somes to lysosomes are abnormal. Alterations in several autophagy processes have also been described in LSDs. Thus, deregulated mitophagy, which results in the accu mulation of damaged mitochondria, occurs in LSDs, leading to major inflammatory consequences in specific tissues 67, 72 . Perturbations in mitochondrial dynamics are frequently observed, which have been linked to the increased production of reactive oxygen species, ATP production and Ca 2+ imbalance. In LSDs, reduced macro autophagy activity (with a decreased autophagic flux) rather than hyperactive autophagy processes, as seen in numerous autoimmune diseases, seems to be responsible for the accumulation of non degraded cytoplasmic pro teins such as α synuclein, huntingtin (HTT) and others 73 . Mucolipidosis type IV (TABLe 2) , a disease characterized by severe neurological and ophthalmological abnormal ities, is caused by mutations in the MCOLN1 gene and is inherited in an autosomal recessive manner. This gene encodes a non selective cation channel, mucolipin 1, which has recently been shown to be required for effi cient fusion of both late endosomes and autophagosomes with lysosomes 74, 75 . Impaired autophagosome degrada tion results in the accumulation of autophagosomes in LSDs 76 . Microautophagy processes that do not involve de novo synthesis of nascent vacuoles also appear to be impaired in LSDs, and were notably revealed in primary myoblasts from patients with the muscle wasting con dition Pompe disease 77 . Finally, defective CMA compo nents, such as LAMP2A, could also lead to lysosomal dysfunction. For example, mutations in the LAMP2 gene have been claimed to cause Danon disease (inherited in an X linked dominant pattern) 51 . Further investigations are needed to support this assertion.

Lysosomes are involved in pathways central to the immune system, including the degradation of intra cellular and extracellular material, plasma membrane repair, cell death signalling, cell homeostasis and death. Although the direct involvement of lysosomes in immunity is far from fully understood, it has long been expected that lysosome dysfunction will have a major impact in immune diseases (TABLe 2) . Strikingly, however, this field has not been extensively explored. However, elevated levels of lysosomal enzyme activity have been reported to occur in several autoimmune diseases, such as RA, systemic lupus erythematosus (SLE), dermatomyositis and psoriasis 3, 14, 17, 18, [20] [21] [22] [23] .

As discussed, autophagosomes formed during the autophagy process must fuse with lysosomes to generate Mitophagy A key process that selectively disrupts damaged mitochondria by autolysosomal degradation, preventing excessive reactive oxygen species and activation of cell death.

(HTT). Discovered in 1993, HTT is a protein of 348 kDa that is widely expressed within the central nervous system. its structure has been elucidated recently by cryo-electron microscopy. The protein is essential for embryonic development and neurogenesis. it is involved in transcription, vesicle transport, protein trafficking, endocytosis and autophagy.

(SLe). A chronic, relapsingremitting autoinflammatory syndrome that has multiple and heterogeneous symptoms, including arthralgia, swollen joints, fever, fatigue, chest pain, kidney inflammation, cardiovascular disease and neuropsychiatric complications. its aetiology is mostly unknown. Reduction of α-d-mannosidases causes reduced lysosomal breakdown of mannose-based oligosaccharides in many tissues 47 Inherited LSD characterized by immune deficiency (susceptibility to infections including pulmonary infections), facial and skeletal abnormalities, hearing impairment and intellectual deficit 47 Fabry disease α-Galactosidase Reduced lysosomal metabolism of α-galactosyl lipids, globotriaosylceramides, causes vascular diseases (cardio, cerebro and renal diseases) in patients 45, 46 Gaucher disease (types 1, 2 and 3) β-GCase Accumulation of glucosylceramides in leukocytes (especially in macrophages) leads to abnormalities in the visceral organs (type 1) and neurological defects in both children and adults (types 2 and 3) 45, 46 GM1 gangliosidosis β-Galactosidase Abnormal lysosomal storage of GM1-ganglioside (oligosaccharides) causes skeletal manifestations and neurological impairment in humans 45, 46 Krabbe disease (globoid cell leukodystrophy)

Defects in the galactocerebrosidase provoke accumulation of galactosylceramide and galactosylsphingosine (psychosine). Patients' brain histology shows myelin loss, neuroinflammation and axonal degeneration 48

Arylsulfatase A or saposin-B (activator protein; rare cases)

Defects in the enzymes lead to the accumulation of sulfogalactosylceramide in major organs. It affects the different age groups of humans with development signs and symptoms of the disease 45, 46 Mucopolysaccharidoses Enzymes involved in mucopolysaccharide catabolism

Accumulation of mucopolysaccharides within lysosomes leads to skeletal and joint abnormalities in humans 45, 46 Multiple sulfatase deficiency SUMF1 (formylglycinegenerating enzyme needed to activate sulfatases)

Abnormal accumulation of multiple, including sulfated, glycosaminoglycans causes neurodegeneration and psychomotor retardation in humans 49 Pompe disease α-Glucosidase Accumulated undegraded glycogen in the muscles and peripheral nerves was observed in humans 45, 46 Sandhoff disease β-Hexosaminidase A and B Enzyme defects cause GM2-ganglioside accumulation in lysosomes, which induces nervous system damage in humans 45, 46 Mucolipidosis (type II and III) N-acetyl glucosamine phosphoryl transferase α/β

Enzyme deficiency results in accumulation of unphosphorylated glycoproteins, which causes motor function and neurological disorders in humans 45 Mucolipidosis IV Mucolipin-I Defects in this lysosomal membrane protein (Ca 2+ channel) cause accumulation of mucopolysaccharides and lipids, thereby resulting in hepatosplenomegaly , dysmorphic features and neurological disorders in humans 45 Cystinosis Cystinosin (cysteine transporter) Defects in this lysosomal transporter, cystinosin, cause accumulation of cystine in different organs, first in kidneys and later in other organs in humans 45, 46 Danon disease L AMP2 Defects in L AMP2 (especially L AMP2B) cause accumulation of glycogen and other autophagic components in cardiomyocytes of humans, which results in cardiac diseases 50 L AMP2B is highly expressed in the brain, cardiac and skeletal muscles 51 Defective autophagy processes observed in SGs of MRL/lpr mice 56 Crohn's disease Abnormal lysosomal pH Deregulation of proton-sensing G protein-coupled receptor (GPR65) was observed in both mice and human 57 www.nature.com/nrd peptide epitopes for further processing, clear possibly deleterious apoptotic debris, fuel the amino acid pool and produce energy (Fig. 1 ). Any deviation in this com plex processing will affect crucial immune cell functions, such as the control of cytokine release, autoimmune cell anergy and programmed cell death of type I (apop tosis) and type II (autophagy). Secretory lysosomes regulate the release of both pro inflammatory and anti inflammatory cytokines, in a process that is depen dent on the type of stimulation. In addition, lysosomes degrade glucocorticoid receptors, which are essential to bind glucocorticoids, although the reasons are not known 78 . In this complex system, lysosomes execute anti inflammatory action via the phospholipase A2 and cyclooxygenase2 pathways, and also induce inflam mation through the IL1β-caspase1 pathway. In both conditions (pro inflammatory and anti inflammatory), lysosomes act as indirect precursors for autoimmunity. However, induction and suppression of inflammatory signals are stimulus dependent 78 . Lysosomal cathepsins have a central role in degrading biological macromolecules in the lysosomes and in the immune response. There are approximately 12 members in this large protease family, most of which are endo peptidases that can cleave peptide bonds of their protein substrates 79, 80 . Cathepsins A and G are serine proteases, cathepsins D and E are aspartic proteases and cathep sins B, C, F, H, K, L, O, S, V, X and W are cysteine pro teases. For example, cathepsin S is responsible for the degradation of antigens (and autoantigens) in antigen presenting cells (dendritic cells, macrophages and B cells), and is therefore involved at an upstream level in the presentation of MHCII-(auto)antigenic peptide complexes to CD4 + T cells 81 . Cathepsin L preferentially cleaves peptide bonds with aromatic residues in the P2 position and hydrophobic residues in the P3 position. It is central in antigen processing, bone resorption, tumour invasion and metastasis, and turnover of intra cellular and secreted proteins involved in growth regu lation. Cathepsin L deficient mice display less adipose tissue, lower serum glucose and insulin levels, more insulin receptor subunits, more glucose transporter type 4 and more fibronectin than wild type controls 82 . Cathepsin G is primarily known for its function in killing and digestion of engulfed pathogens 83 . It is also involved in connective tissue remodelling at sites of inflammation 84 . Anti neutrophil cytoplasmic antibodies reacting with cathepsin G have been identified in some patients with SLE 85 .

Abnormal antigen processing and presentation is known to be one of the upstream events that perturb immune responses in SLE 86 . Because this process is mediated through lysosomes, it was rational to speculate that lyso somal functions could be altered in lupus. Interestingly, hypotheses were raised in the 1960s on the 'lysosomal fragility' in lupus, but without much further pursuit 87 . The composition and fluidity of the lysosomal mem brane are effectively crucial in the regulation of lyso somal fusion with other vesicular organelles and for lysosomal uptake of macromolecules. The integrity of the lysosomal membrane also ensures the prevention of release of lysosomal enzymes into the cytoplasm. Some lysosomal enzymes released from 'fragile' lysosomes were regarded potentially harmful in lupus 88 .

Autoimmune diseases (cont.) Lysosomes are abnormal in splenic B cells from Fas deficient Murphy Roths Large (MRL)/lpr mice, a mouse model of lupus, compared with B cells from healthy CBA/J mice 89 . TFEB expression was increased, indicating an enhanced biogenesis of lysosomes, and the lysosomal volume was raised. The expression levels of LAMP1 and cathepsin D were also increased. These results reinforce previous data showing that the expres sion and activity of some lysosomal enzymes (such as cathepsins S, L and B) that play important roles in antigen processing are altered in lupus and other autoimmune diseases 90, 91 .

Substantial variations of the acidic endo lysosomal pH also occur in MRL/lpr mice, being raised by 2 pH units in splenic B cells 53, 92 . This pH change could dramat ically influence the activity of soluble lysosomal hydro lases (such as cathepsins) as well as lysosomal membrane proteins (such as LAMPs) that are critical for lysosome activity. pH may also affect the elimination of immune complexes that accumulate in lupus as a result of deficits in complement, lower expression of scavenger receptors, increased expression of Fcγ receptors and other rea sons 93 . These immune complexes, which contain non selective IgG antibodies or autoantibodies associated with autoantigen (including some apoptotic debris), can initiate inflammation of tissues once deposited (for example, in the kidneys and the skin) and generate a cas cade of deleterious effects, such as the release of harmful cytokines and chemokines 54 .

Recent studies have highlighted the key role of mam malian target of rapamycin complex 2 (mTORC2) in the disruption of lysosome acidification that occurs in this process 94 . In normal conditions, the regulation of lyso somal acidification requires cleavage of the RAB small GTPase RAB39a, occurring on the surface of phago cytic vesicles by locally activated caspase1 94 . This finely regu lated process requires the association of cofilin with actin that surrounds the vesicle and recruits caspase11, which then activates caspase1 (ReF. 94 ). In lupus prone macro phages, chronically active mTORC2 enhances cofilin phosphorylation, thereby hampering its association with actin and affecting the downstream cascade of events leading to the appropriate acidification of lysosomes 94 . The importance of mTORC1 and mTORC2 has been established earlier in lupus T cells, and in particular, in this context, mTORC1 activity was increased whereas mTORC2 activity was reduced 95 .

In addition, lysosomal cathepsin K was seen to contrib ute to the pathological events that develop in Fas lpr mice, another model of lupus disease, in part through its activity in TLR7 proteolytic processing and subsequent effects on regulatory T cells. Cathepsin K deficiency in Fas lpr mice reduced all kidney pathological manifesta tions (glomerulus and tubulointerstitial scores, glo merulus complement C3 fraction and IgG deposition, chemokine expression and macrophage infiltration) and decreased the levels of potentially pathogenic serum autoantibodies 96 .

In line with these internal alterations of lysosomes, notably those related to cathepsin functioning, deregu lation of autophagy has been reported to contribute to lupus pathology 92,97-100 . Autophagy failures have been described in the lymphocytes of MRL/lpr mice and (NZBxNZW)F1 mice 56, 92, 97, 101 (two spontaneous murine models of systemic autoimmunity of distinct genetic ori gins and that display different MHC haplotypes) as well as in T and B lymphocytes of patients with SLE 97,98,100 . Murine and human T cells from the peripheral blood showed a significant accumulation of autophagic vac uoles compared with normal 97 . The underlying reasons for the dysfunctions in autophagy observed in lupus are not clearly understood, but several indepen dent investigations have identified risk loci spanning autophagy linked genes in patients with lupus 102-106 .

Recent studies have demonstrated an increase in the level of macroautophagy in salivary gland T lymphocytes and in tears and conjunctival epithelial cells of patients with primary Sjögren's syndrome (SjS) 107, 108 . Alteration of CMA activity was also recently found to occur in the salivary glands of MRL/lpr mice that develop a second ary SjS like disease 56 . Lysosomes, which as discussed are mechanistically involved at the downstream level of both macroautophagy and CMA, were found to be altered in salivary glands. Flow cytometry analyses revealed that the mean pH of acidic vesicles in MRL/ lpr salivary glands was significantly higher compared with those in mouse control glands and the ATP con tent was significantly diminished in MRL/lpr salivary gland cells 56 . Furthermore, amounts of several leukocyte glycosidases and proteases were revealed to be increased in leukocytes of patients with SjS in comparison with healthy controls 55 . Notably, raised levels of the lyso somal enzymes glucosidase, β glucuronidase and dipeptidyl peptidase I are involved in the tissue injury in SjS 55 . Increased expression of lacrimal gland cathepsin S was also reported, which may have application as a diag nostic tool in SjS 91 . Two members of the RAS oncogene family, RAB3D and RAB27, were found to be implicated in the regulation of cathepsin S secretion levels in SjS 109 . In vitro studies on lacrimal gland acinar cells suggested further that secreted IFNγ from acinar cells increases cathepsin S expression and that IFNγ stimulated the MHCII mediated antigen presentation in ocular pathogenesis of SjS 110 .

Lysosomal cathepsins have important roles in the induc tion and diagnosis of RA, and levels of several cathepsins (B, D, G, K, L and S) that are present in the serum and synovial fluid of patients have been proposed as a basis for RA diagnosis [111] [112] [113] [114] [115] [116] . Cathepsin S and cathepsin L are highly expressed in synovial macrophages and thymic cortical cells. They each exert essential roles in the posi tive selection of T cells and antigen presentation, respec tively, and participate in the local inflammation and matrix degradation that occurs in joints 116 . Cathepsin B is involved in collagen degradation, which leads to joint destruction in RA 112, 117 . Expression of cathepsin G, which participates in joint inflammation through its chemoattractant activity, has been shown to be raised in the synovial fluid of patients with RA when compared with individuals with osteoarthritis 115 . Autoantibodies www.nature.com/nrd reacting with cathepsin G were also identified in patients with RA 85 . Compared with patients with osteoarthritis, cathepsin K expression was found to be elevated in RA 113 , and genetic deletion of this particular cathepsin was shown to reduce inflammation and bone erosion in RA conditions via TLR mediation 118 .

Neurological autoimmune diseases MS, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), neuromyelitis optica and neuropsychiatric lupus are neurological diseases induced by abnormal auto immunity 62, [119] [120] [121] [122] [123] . Neurological autoimmunity against various proteins, such as myelin in MS or N methyl d aspartate receptor in neuropsychiatric lupus 62, 123, 124 , can affect various structures within the CNS and peripheral nervous system, with diverse consequences. Although the exact cause of amyotrophic lateral sclerosis (ALS) still remains unknown, studies support the existence of auto immune mechanisms, and ALS is therefore also included in this section. Indeed, autoantibodies against ganglio side GM1 and GD1a, sulfoglucuronylparagloboside, neurofilament proteins, FAS/CD95 and voltage gated Ca 2+ channels have all been reported in patients with ALS (reviewed elsewhere 125 ) .

In general, the origin of the breakdown in immune tolerance that occurs in this set of neurological diseases is not known. Only recently have investigations discov ered that autophagy processes are altered in some of these diseases 59, 62, [126] [127] [128] [129] [130] . In MS and in experimental auto immune encephalomyelitis, an experimental model of MS, upregulation of the protein kinase mTOR has been described, and treatment with rapamycin/sirolimus (an immunosuppressant that inhibits mTOR and conse quently stimulates macroautophagy) ameliorates some clinical and histological signs of the disease 131 . Increased levels of macroautophagy markers were measured in the blood and brain of patients with MS 122,132 . However, impaired macroautophagy was found in the spinal cord of experimental autoimmune encephalomyelitis mice 133 . In a rat model mimicking human CIDP, both macro autophagy and CMA processes were found to be hyper activated in lymphatic system cells and non neuronal cells (sciatic nerves) of peripheral nervous system cells 59 . In ALS, current data are conflicted 62 . Some data suggest an activation of macroautophagy processes with an accumulation of autophagosomes in brain tissues of patients with ALS, or an increase of autophagic vacuoles, aggregated ubiquitin and SOD1 proteins associated with MAP1LC3B II in motor neurons of mice developing an ALS like disease 134, 135 . In contrast, other data suggest a reduction of autophagy activity 136, 137 . Mutations in SQSTM1, valosin containing protein, dynactin (a pro tein complex that activates the dynein motor protein, enabling intracellular transport) and RAB7 (a member of small GTPases that is important in the process of endosomes and autophagosomes maturation) have also been described in ALS [138] [139] [140] [141] . Further studies are required to better understand the type and extent of autophagy dysfunction in this family of complex diseases.

There are only a few published studies on lyso somal dys function in neurological autoimmune diseases (TABLe 2) .

These notably include lysosome fragility, which was observed in patients with MS in the white matter of cerebral tissue, an area of the CNS that is mainly made up of myelinated axons 142 . Lysosome fragility was also suspected in SLE (see above) and other rheumatic auto immune diseases, albeit in other organs 53, 58, 92 . As noted above, significant variations in lysosomal pH have been measured in autoimmune conditions such as lupus and SjS, but to our knowledge such studies conducted in the brain or elements of the peripheral nervous system of patients or animal models with neurological autoimmune diseases have not been published 78 .

In CIDP, it has been shown that Schwann cells dedif ferentiate into immature states and that these dediffer entiated cells activate lysosomal and proteasomal protein degradation systems 143, 144 . Based on these observations, Schwann cells have been claimed to actively participate in demyelinating processes via this dedifferentiation process, but the mechanism involved remains unde fined 145 . In the rat model of CIDP mentioned above, it was shown that LAMP2A expression was drastically increased in the sciatic nerve macrophages and reduced macroautophagy was observed in Schwann cells and macrophages 59 .

In MS, studies conducted on white matter demon strated that lysosomes are involved in myelin sheath degeneration as well as in the fragmented protein forma tion. Lysosomal swelling was observed near the degen erated materials of astrocytes 146 , and an accumulation of lipids was found 60 . It has been hypothesized that lyso somal swelling/permeabilization might cause the release of hydrolases in the cytosol, where they affect native proteins 147 .

In ALS, patients also show dysfunctions in the endo/ lysosomal pathways, which affect both lower and upper motor neurons (TABLe 2) . Cathepsin B was particularly found to be involved in the motor neuron degeneration, whereas cathepsins H, L and D were not significantly affected 148 . A cDNA microarray analysis on post mortem spinal cord specimens of four sporadic patients with ALS revealed major changes in the expression of mRNA in 60 genes including increased expression of cathepsins B and D 149 . Several disease causing mutations in genes related to autophagy have been identified, such as SOD1, TDP643, FUS, UBQLN2, OPTN, SQSTM1 and C9orf72 (ReFS 61,150 ), but none of them code for lyso somal proteins. So, a crucial remaining issue is to clearly determine whether the lysosomal abnormalities that are observed are linked to intrinsic defaults of lysosomes or result from upstream dysregulation in autophagosome formation and fusion 61, 62, 151 .

Insufficient clearance of neurotoxic proteins by the autophagy-lysosomal network has been implicated in numerous neurodegenerative disorders 152 . In disorders such as AD, Huntington disease (HD) and PD, modi fied or misfolded proteins abnormally accumulate in specific regions of the brain. Accumulation of aggregated proteins is also seen in ALS (see above). These abnor mal proteins form deposits in intracellular inclusions or extracellular aggregates, which are characteristic for

Caused by antibodies targeting the muscle acetylcholine receptor or other neuromuscular junction proteins such as musclespecific kinase. These antibodies compromise communication between nerves and muscles, leading to muscular weakness and fatigue.

Chronic inflammatory demyelinating polyneuropathy (CiDP). A progressive autoimmune disorder in which peripheral nerves (roots and trunks) and brachial plexuses are damaged owing to demyelination. it causes muscle weakness, sensory loss and reduced reflexes.

Also known as Devic's syndrome, this disease is characterized by an inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis). Antibodies reacting with aquaporin-4 water channels in the brains of patients are implicated in neuromyelitis optica.

(ALS). Also known as motor neuron disease, this disease generally starts with muscle twitching and weakness in a limb, or slurred speech. it can affect control of the muscles needed to move, speak, eat and breathe, and can be fatal.

NATure revIewS | Drug DiSCOvErY each disease [153] [154] [155] . Although there has been substantial research in this field, it is still unclear why sophisti cated 'quality control' systems, such as the lysosomeautophagosome system in particular, fail in certain cir cumstances to protect the brain against such protein accumulation 156 .

In AD, one of the most common neurodegenerative disorders, some alterations in the endo/lysosomal path ways have been described (reviewed elsewhere 157, 158 ). The amyloid precursor protein (APP) is cleaved by β and γ secretases into amyloid β peptide (Aβ) fragments, particularly Aβ40 and Aβ42 (ReF. 159 ). These fragments are found in the amyloid plaques that are one of the hallmarks of AD (the other being neurofibrillary tangles containing phosphorylated tau), and have been widely considered to have an important role in AD pathogen esis 159, 160 . Cell based experiments have demonstrated that lysosomal cathepsins have a role in the generation of Aβ peptides (through cathepsins D and E) and the degrada tion of Aβ peptides (by cathepsin B) 161 . Lysosomal dys function has been observed in patients with AD 162, 163 , and accumulation of the Aβ42 fragment in neuronal cells was shown to lead to lysosomal membrane alter ations, which cause neuronal cell death 63 . In this context, it is noteworthy that inhibition of cathepsin D, which is involved in the lysosomal dysfunction and notably in the cleavage of the tau protein into tangle like fragments, diminishes its hyperphosphorylation in the brain of patients with AD 164 . In addition, patients with AD with an inherited form of the disease may carry mutations in the presenilin proteins (PSEN1 and PSEN2), APP or apolipoprotein E, resulting in increased production of the longer form of the Aβ fragment (reviewed else where 165 ). Mutation of PSEN1, for instance, leads to direct disruption of the lysosomal acidification due to impaired delivery of the V0A1 subunit of v ATPase, a proton pump responsible for controlling the intracellu lar and extracellular pH of cells. The acidification defi cit causes excessive release of lysosomal Ca 2+ through TRPML1 channels, which has numerous deleterious effects 166 . These findings strongly support the hypothesis that dysfunction of endo/lysosomal pathways is pivotal in AD.

Approximately 15% of patients with PD have a family history of the disorder, although the underlying molec ular mechanisms remain unclear. In the context of lysosomal dysfunction, it is notable that the most com mon of the known PD genetic mutations are in GBA1 (encoding the lysosomal β GCAse) -the same gene that underlies Gaucher disease -which are present in up to 10% of patients with PD in the United States 167 . GBA1 mutations are also associated with dementia with Lewy bodies 167 . Several other genes linked to PD are directly or indirectly related to the endo/lysosomal machinery, such as mutations in SNCA (coding for α synuclein) 63, 168 . A hallmark of PD is the presence in neurons of protein inclusions called Lewy bodies, which are mainly com posed of fibrillar α synuclein. The α synuclein protein is normally degraded by the lysosomes through the CMA pathway, but macro aggregates of α synuclein mutants, which display a longer half life compared with the non aggregated wild type protein, are not degraded by this pathway and, rather, would be degraded via the macro autophagy pathway [169] [170] [171] [172] . It was further shown that the mutant proteins bind to LAMP2A and inhibit the translocation of other substrates and, therefore, their final degradation 170 . Biochemical analyses suggest that α synuclein is mainly degraded by lysosomal proteases and notably by cathepsin D, rather than by non lysosomal proteases (for example, calpain I) 173, 174 . Accumulation of α synuclein was observed in cathepsin D deficient mice, whereas, conversely, the accumulation of α synuclein aggregates was reduced in transgenic mice that over expressed this cathepsin, resulting in protection of dopaminergic neuronal cells from damage 175 .

HD is a rare autosomal dominant neurodegener ative disease caused by an aberrant expansion of CAG trinucleotide repeats within exon 1 of the HTT gene, which results in the production of aggregation prone HTT mutants (mHTT) that are detrimental to neu rons 176, 177 . Whereas HTT has a protective role against neuronal apoptosis, accumulation of mHTT, however, induces pathophysiological consequences including lysosomal and autophagy dysfunctions. Thus, mHTT perturbs post Golgi trafficking to lysosomal compart ments by delocalizing the optineurin/RAB8 complex, which, in turn, affects lysosomal function 177 . Excessive mHTT induces accumulation of clathrin adaptor com plex 1 in the Golgi and an increase of clathrin coated vesicles in the vicinity of Golgi cisternae 177 . The activity of several cathepsins such as B, D, E, L and Z has also been linked to HD 63, 80, 174, [177] [178] [179] . Cathepsin D is respon sible for full degradation of HTT but is less efficient at degrading mHTT, which is processed by cathepsin L 180, 181 . Cathepsin Z also cleaves HTT and elongated poly glutamine tracts 182, 183 . Thus, lysosomal modulators acting on cathepsin activity might have beneficial effects in the treatment of HD. Notably, hyperexpression of cathepsin D (and cathepsin B) was shown to protect primary neurons against mHTT toxicity 179 . Alterations in macro autophagy, mitophagy and CMA have also been implicated in HD 184, 185 . CMA activity was increased in response to macroautophagy failure in the early stages of HD 186 , a result supported by the findings that HSPA8 and LAMP2A have important roles in the clearance of HTT 187 and that shRNA mediated silencing of LAMP2A increased the aggregation of mHTT 188 . Other studies focusing on the HTT secretory pathway revealed that mHTT secretion is mediated by the Ca 2+ dependent lyso somal exostosis mechanism via the synaptotagmin 7 sensor in neuro2A cells 189 . The extracellular release of mHTT was efficiently inhibited by the phosphoinositide 3 kinase and sphingomyelinase inhibitors Ly294002 and GW4869. HD dependent perinuclear localization of lysosomes was also demonstrated 190 .

Increasing evidence thus implicates lysosomal (and autophagy) dysfunction in the pathogenesis of neuro degenerative disorders 62, 63, 127, 128, 130, 191, 192 . TFEB has received particular attention in this regard [193] [194] [195] , with recent data suggesting that TFEB is selectively lost in patients with AD (as well as ALS) 196 . Increasing TFEB activity might therefore prevent neuronal death and restore neuro nal function in certain neurodegenerative diseases, including PD 194 .

A major microtubuleassociated protein of a mature neuron. Hyperphosphorylated tau accumulates with ubiquitin in ageing neurons as the neurofibrillary tangles that were identified in numerous neurodegenerative diseases called tauopathies that include Alzheimer disease.

Lysosomes as therapeutic targets Given the evidence discussed above, the various lyso somal pathways and their components could represent potential pharmacological targets for a wide range of diseases. When considering lysosomes as targets, it is important to note the need for specificity; that is, agents that will not target all lysosomes, but will specifically tar get those lysosomes/lysosomal proteins that are defective in certain organs, tissues or cells. In addition, inhibitors or activators of lysosomal components may be required, depending on the disease context.

There has been considerable interest in therapeuti cally targeting different autophagy pathways, including lysosome dependent pathways, and progress in the discovery and development of small molecules and biologics that target these processes has been reviewed extensively 11, [119] [120] [121] [122] 197, 198 . However, very few therapies that specifically target lysosomal components have so far been generated and found to be effective in clinical trials, with one general exception -the development of ERTs and small molecule drugs for LSDs (Box 1). This topic has recently been comprehensively reviewed 46 and so will not be discussed in depth here.

It is important to target lysosomes and not the whole autophagy process for several reasons. First, regard ing safety, the integral role of lysosomes in several key physio logical processes means that therapeutic windows for pharmacological intervention with unacceptable side effects may be limited. For example, azithromycin, an antibiotic with anti inflammatory properties that is used in the treatment of patients with chronic inflammatory lung diseases such as cystic fibrosis, was found to block autophagy in macrophages, inhibiting intracellular kill ing of mycobacteria within them and, thereby, increasing the risk of mycobacterial infection 204 . Second, in some diseases, autophagy may be enhanced in certain tissues or organs but compromised in others, for example in the spleen and salivary glands of MRL/lpr mice 56 . This phe nomenon makes it highly challenging to identify a single drug able to correct a failure, unless a cell specific target ing molecule could be incorporated into the autophagy activator/inhibitor to enable tissue specificity 205 . Again, the precise targeting of lysosomes in specialized cells may circumvent the complexity of dysregulation mecha nisms of autophagy processes in pathophysiological settings 14, 56, 206, 207 .

As indicated, the current arsenal of lysosome specific targeted drugs is small. In fact, many drugs claimed to target lysosomal components have also been found to be capable of interacting with several non lysosomal receptors, limiting their efficacy and safety 12 . One exam ple is provided by chloroquine (CQ), a 4aminoquino line compound, and its derivative hydroxychloroquine (HCQ), which are widely prescribed to patients with rheumatic diseases, and historically also for the prophy laxis and treatment of malaria (Fig. 3) . CQ and HCQ are lysosomotropic agents and as such they raise intralyso somal pH, thereby affecting overall lysosomal function and impairing autophagic protein degradation (Fig. 2) . Although the mechanism of action of these agents is not fully elucidated, it is well established that CQ and HCQ display pleiotropic activity [208] [209] [210] and have impor tant deleterious properties. In certain settings, they have been claimed to operate by interacting directly with TLR ligands and not through an effect on the lysosomal pH, for example 211 . Toxicity of CQ/HCQ, in particular in the eye (cornea and macula) and the occurrence of cardio myopathies 212 , remains a major limitation. The observed ocular toxicity is related to the total cumulative dose rather than the daily dose; therefore, it becomes a serious potential problem in the cases of long term use. Several HCQ analogues and mimics have been designed that aim to retain the therapeutic activity without secondary effects 213, 214 .

Furthermore, most, if not all, of the small molecules that have so far been identified and investigated as mod ulators of autophagy and/or lysosomal functions exhibit complex pleiotropic properties affecting the overall function of lysosomes, and also different autophagy pathways (for example, mTOR dependent and mTOR independent pathways), as well as other quality control mechanisms that affect the cell life/death balance. As dis cussed below, several widely used molecules exert dual, sometimes opposite, effects on upstream and down stream molecular events of the autophagy-lysosomal network.

Several robust assays to characterize autophagy acti vators and inhibitors, as well as lysosomal effectors, are currently available and validated (TABLe 3) . However, each assay has inherent biases, and so it is necessary to use several independent, in vitro and in vivo approaches to ascertain the reactivity and specificity of novel molecules able to modulate these pathways (Box 2).

In this regard, the tremendous work in recent years to establish international guidelines for standardizing research in autophagy -and, in particular, to propose relevant methodologies for monitoring autophagy that are accepted by the whole community -is unique 231, 232 . A better definition of terms and concepts has also Box 1 | Enzyme replacement therapies for lysosomal storage disorders enzyme replacement therapy (erT) for lysosomal storage disorders (lSD) involves administration of a functional version of the defective enzyme in the particular lSD. Following administration, the enzyme is delivered to the target cells (typically mediated by mannose or mannose-6-phosphate receptors), where it breaks down its substrate in lysosomes, thereby ameliorating the lSD 46 .

The approach was pioneered with the use of glucocerebrosidase (Gcase) purified from placentae in the 1980s to treat patients with Gaucher disease, and a recombinant version of Gcase was then introduced in the 1990s 199 . Following the success of this approach in treating Gaucher disease, other recombinant enzymes have been approved for other lSDs, including Fabry disease, mucopolysaccharidosis (mPS) I, mPS II, mPS vI and Pompe disease (TABLe 1) , and many further erTs for other lSDs are in clinical trials 200 .

Although erT has provided an effective treatment for patients with some lSDs, it has limitations. recombinant enzymes administered by intravenous injection are not able to cross the blood-brain barrier, and so are not effective for central nervous system manifestations of lSDs 201 . low expression of the receptors that mediate delivery on the cell surface of target cells can also be a challenge for the effectiveness of erT for some lSDs 46 . For example, in Pompe disease, the level of expression of mannose receptors on skeletal muscle cells is low, necessitating high doses of erT to achieve a therapeutic effect 202 . Numerous developments are being studied to address such limitations, with a focus on enzyme modifications that enable better access of enzymes to their receptors and on nanomaterials that enable safe and efficient delivery of enzymes via intra-cerebroventricular/intrathecal administration 10, 46, 200, 203 . been adopted by the community, leading to much eas ier understanding between researchers worldwide 233 . These guidelines and definitions should be used by investigators evaluating new molecules designed to selectively target key steps of autophagy or developing new high throughput screening methods for autophagy modulating pharmacological molecules. However, even the more sophisticated and detailed assays will not reca pitulate the full complexity of integrated living systems, which can only be established in clinical trials.

The pipeline of specific agonists and antagonists of autophagic activity is currently small, particularly for CMA (TABLeS 4,5; FigS 2,3). However, high throughput screening programmes to identify such small molecules are ongoing, which should yield additional therapeutic targets and useful tools. Small molecules that specifically target lysosomes are even rarer (TABLe 4; Fig. 2 ). Small molecule drugs developed specifically for particular LSDs, including substrate reduction therapies and small molecule chaperones, have reached the market, but other small molecule candidates for more common diseases are at an earlier stage of development. These molecules that more specifically act on lysosomes, some of which have been discovered by high throughput screening, mostly target LAMP2A, various lysosomal enzymes such as cathepsins, acid sphingomyelinase, α galactosidase A and acid β glucocerebrosidase, and chaperones such as HSPA8 and β Nacetyl hexosaminidase. Although not solely present in lysosomes, v ATPase, a proton pump responsible for controlling the intracellular and extracel lular pH of cells, and TRPML1, a cation channel located within endosomal and lysosomal membranes, are also pertinent targets.

Below and in TABLe 4, we summarize the availabil ity of pharmacological tool compounds and progress in drug development, where applicable, for each broad target class.

In addition to ERTs for LSDs (Box 1), drug discovery programmes have also focused on alter native small molecule based approaches, which may be particularly relevant for LSDs that affect the CNS, due to the lack of blood-brain barrier penetration by ERTs 283 .

Small molecules used in substrate reduction thera pies prevent the accumulation of substrates of the defec tive enzymes in LSDs by inhibiting enzymes involved in substrate production 284 . Miglustat was the first such drug to be approved in the early 2000s by the US Food and Drug Administration and the European Medicines Agency for Gaucher disease and in 2009 for Niemann-Pick disease type C in Europe. This iminosugar inhibits glucosylceramide synthase (GCS), which catalyses the initial step in formation of many glycosphingolipids. Within cells, glycosphingolipids tend to localize to the outer leaflet of the plasma membrane; they cycle within the cell through endocytic pathways that involve the lysosome. Inhibition of GCS therefore reduces the deleterious accumulation of glycosphingolipids within lysosomes with potential therapeutic benefits in dis eases like LSDs. Miglustat also inhibits disaccharidases in the gastrointestinal tract, resulting in diarrhoea as a side effect 285 . Eliglustat, another GCS inhibitor that does not penetrate the CNS, was also approved for Gaucher disease in 2014. Other GCS inhibitors in clinical devel opment include lucerastat, a miglustat analogue with an improved safety profile that is currently in a phase III trial for Fabry disease (FD) 236, 286 , and ibiglustat, which penetrates the CNS. The latter is in clinical development for FD (phase II), for Gaucher disease type 3 (phase II) and for patients with PD who carry a mutation in GBA (phase II). Recent findings generated in a small number of patients have suggested a possible link between PD and FD 287 , which also exists between patients with PD and Gaucher disease who have GBA mutations (see above). Finally, genistein, a pleotropic natural product that inhibits kinases involved in the regulation of proteo glycan biosynthesis and also affects TFEB function, is in a phase III trial for Sanfilippo syndrome 288 .

Substrate mimetics that inhibit lysosomal enzymes have also been found to stabilize mutated enzymes in LSDs, thereby leading to restoration of some enzyme activity when suitable subinhibitory concentrations are used, as the enzyme remains stable and functional after dissociation of the inhibitor 46, 283 . The pioneering exam ple of this approach is migalastat, described above, that binds to the active site of α galactosidase A, which is mutated in FD, and stabilizes the mutant enzyme. Other examples of this strategy include afegostat in Gaucher disease (which failed in a phase II clinical trial in 2009 due to lack of efficacy), pyrimethamine in Sandhoff disease and Tay-Sachs disease, and ambroxol in Gaucher disease with neurological symptoms (TABLe 4). Agents that are at earlier developmental stages include N octylβ valienamine, a competitive inhibitor of βglucosidase, for Gaucher disease; N acetylcysteine for Pompe disease; α lobeline, 3,4,7trihydroxyisoflavone and azasugar in Krabbe disease; and N octyl4epi β valienamine and 5N,6S(N′butyliminomethylidene) 6thio1deoxygalactonojirimycin indicated in GM1 gangliosidosis 289 . The chemical structures of these pharmacological chaperones have been described recently 290, 291 . Finally, an alternative strategy for stabiliz ing mutant enzymes, by binding away from the active site, is also being investigated. A promising example of this approach is NCGC607, a non inhibitory small molecule chaperone of GCase discovered by screening for molecules that improved the activity of the mutant enzyme 46, 250 . Treatment with NCGC607 reduced lyso somal substrate storage and αsynuclein levels in dopamin ergic neurons derived from induced pluripotent stem cells from patients with Gaucher disease with parkinson ism 46, 250 . Further testing of NCGC607 in patients with PD and GBA mutations is awaited. Although promis ing, conflicting viewpoints still remain on the strength of such small molecule based approaches, primarily because these compounds bind to the catalytic site of enzymes, which may be a risk at high concentrations if they inhibit rather than increase activity 291, 292 . More clinical trials are therefore required in order to analyse the robustness of this approach.

Cathepsin modulators. Robust genetic and pharma cological preclinical investigations have consistently showed that regulating cathepsin activity can favour ably improve pathological features in certain auto immune and inflammatory diseases. Inhibitors of several cathepsins (B, D, L, K and S) have been described 174, 293 and their activity has been evaluated in rheumatic auto immune diseases (such as SLE, RA and SjS) and neuro degenerative disorders, notably in AD 294 (TABLe 4) . Selective inhibition of cathepsin S with a potent active site inhibitor known as RO5461111 (Roche) mitigated disease in MRL/lpr lupus prone mice, by reducing prim ing of T and B cells by dendritic cells, and plasma cell generation 262 . Promising data have also been generated in murine models, in the context of diabetic nephrop athy and cardiovascular diseases 295 . Further studies based on cathepsin S inhibitors should evaluate the clinical safety and utility of treating patients affected by autoimmune and inflammatory diseases 295 . Cathepsin K, which is highly expressed by osteoclasts and very effi ciently degrades type I collagen, the major component of the organic bone matrix, is also a potential target for modulating lysosomal dysfunction in some of the dis orders discussed above, such as SLE 96 . Yet further investi gations with selective cathepsin K inhibitors are required to determine whether this targeted strategy might apply in SLE and other inflammatory conditions in which articular manifestations are a major component (RA, ankylosing spondylitis, psoriatic arthritis and others). It should be noted, however, that various cathepsin K inhibitors have been pursued for postmenopausal osteo porosis, including odanacatib (Merck) which reached phase III trials 296 . Although odanacatib was effective, its development was discontinued in 2016 due to an increased risk of stroke in treated patients. Other cathep sin inhibitors and their context of clinical evaluation are listed in TABLe 4. Despite multiple efforts to develop selective pharma cologic cathepsin modulators, important concerns still Fluorescence measurement (flow cytometry or fluorescence microscopy) of cellular staining of acidotropic dyes, such as LysoTracker dyes 92, 215 Simple to use but is not quantitative as stated by the manufacturer; can be adapted to clinical trial settings Western blot and fluorescence imaging of lysosomal markers such as L AMP1, L AMP2 etc. 216, 217 Simple but does not provide information on subcell populations 89 Limited usage in primary cells as they are hard to transfect BSA , bovine serum albumin; L AMP, lysosome-associated membrane protein; N/A , not available; qPCR , quantitative PCR; TFEB, transcription factor EB.

www.nature.com/nrd remain with regard to off target effects due to activity against other cathepsins or towards cathepsins present at non relevant or unwanted sites. Nonetheless, the under lying biology and clinical effects of certain cathepsin inhibitors or activators remain of considerable interest and could guide future therapeutic approaches.

As reported below, v ATPase, a multisubunit ATP driven proton pump, is best known for its role in acidification of endosomes and lysosomes. Regulating the function of v ATPase may impact lyso somal activity and, hence, the acidification of spe cialized cells and diverse signalling pathways, such as autophagy. v ATPase inhibitors like bafilomycin A1 and concanamycin A are non selective compounds (TABLe 4; Fig. 3 ) that inhibit both mammalian and non mammalian v ATPases, which control the lysosomal pH of acidic vesicles via a manner that is not fully under stood (Fig. 2) . Through this mechanism, bafilomycin A1 inhibits autophagic flux by preventing the acidifi cation of endosomes and lysosomes 297 . Bafilomycin and CQ also affect mitochondrial functions, as discovered recently using intact neurons 298 . Benzolactoneenamides (salicylihalamide A, lobatamides and oximidines; 299 than bafilomycin A1 and concanamycin A, but also much less potent. Further investigations into v ATPase regulation of signalling pathways are needed to identify specific and safe molecules that regulate this vital proton pump 300 .

Ion channel modulators. As discussed above, lysosomal ion channels are master elements of lysosome activity and, thereby, of cell homeostasis. In the family of TRP channels, TRML1 is essential, being widely expressed in late endosomes and lysosomes, and preferentially associ ates with LAMP1 in the lysosomal membrane 22, 301, 302 . Genetic mutations leading to inactivation of TRPML1 cause a rare genetic disorder called mucolipidosis type IV (MLIV). Pharmacological activation of TRPML1 ameliorated some lysosomal functions that are clas sically associated with MLIV, NPCs and certain LSDs (TABLeS 2,4; Fig. 2 ). Thus, the small molecule SF22 (Fig. 3) , which was identified in a screen for TRPML3 acti vators, was defined as an activator of both TRPML3 and TRPML1 (ReF. 274 ), and displayed an additive effect in combination with the endogenous activator phospha tidylinositol3,5bisphosphate (PtdIns(3,5)P2) 274, 303 . An analogue of SF22, in which chlorine on the thiophene had been replaced by a methyl group, showed greater efficacy on TRPML1 activation 303, 304 . Another molecule called ML SA1 (FigS 2,3) , acting as a mucolipin synthetic agonist, also showed an additive effect with endogenous PtdIns(3,5)P2 on TRPML1 channels 305 . It is important to note that in neurological diseases, as well as in other indications in which lysosomal acidification is defec tive (see above), interfering with TRML1 may have contraindications.

A central modulator of lysosomes is the lipid kinase FYVE finger containing phosphoinositide kinase (PIKfyve), which converts phosphatidylinositol3phosphate into PtdIns(3,5)P2. The latter regulates Ca 2+ release from the lysosome lumen and is required for acidification by v ATPase. Inactivation of PIKfyve leads to many patho physiological problems including neurodegeneration and immune dysfunction, mostly related to impaired autophagic flux and alteration of lysosomes (trafficking, Ca 2+ transport, biogenesis and swelling) 306 . The small molecule apilimod ( Fig. 3; TABLe 4 ) was originally identi fied as an inhibitor of TLR induced IL12 and IL23, and later found to be a highly specific inhibitor of PIKfyve 276 . Apilimod was evaluated in clinical trials involving sev eral hundred patients with T helper 1 and T helper 17 cell mediated inflammatory diseases such as Crohn's dis ease, RA and psoriasis 277, 278 . It was well tolerated in more than 700 human subjects (normal healthy volunteers and patients with inflammatory disease), but the clinical trials did not meet their primary endpoints and further development was abandoned. Apilimod is currently being evaluated in a clinical trial (NCT02594384) aimed at defining a maximum tolerated dose in patients with B cell non Hodgkin lymphoma and monitoring safety, pharmacokinetics, pharmacodynamics and prelimi nary efficacy 307 . YM201636 is another selective inhibi tor of PIKfyve (TABLe 4; Fig. 3 ). This inhibitor contains a FYVE type zinc finger domain. YM201636 was found to significantly reduce the survival of primary mouse hippo campal neurons in culture and reversibly impair endo somal trafficking in NIH3T3 cells, mimicking the effect produced by depleting PIKfyve with small interfering RNA. It was also found to block retroviral exit by budding

Several parameters have been used to evaluate lysosomal functions (TABLe 2) . Alteration of lysosomal volume is an important sign of lysosomal dysfunction; it has been observed in various diseases, such as autoimmune syndromes, cancers and lysosomal storage diseases 215 . It can be measured by staining cells with acidotropic dyes such as lysoTracker dyes and immunoblot of lysosomal membrane proteins such as lysosome-associated membrane protein 1 (lAmP1). variation of lysosomal volume is often related to changes in lysosomal biogenesis, which can be assessed by the expression level and cellular location of transcription factor eb (TFeb). However, precise determination of lysosomal functions relies on measurement of lysosomal luminal pH and degradation activity. Several fluorescence probes that measure lysosomal pH (TABLe 2) are commercially available. Abnormal lysosomal pH affects lysosomal degradation activity, which can be followed, for example, by detecting the degradation of endocytosed fluorescence DQ-bSA 57 . In complement, the activity of specific enzymes, such as cathepsins b, D and l, can be tested using commercially available kits. other lysosomal parameters can be evaluated to deepen the examination of lysosomal status, including lysosomal membrane stability and integrity and lysosomal ca 2+ ion signalling, for example (TABLe 2) . lysosomal function is essentially linked with autophagy activity as autophagy is a lysosomal-dependent degradation pathway. Thus, a series of methods routinely applied for assessing macroautophagy in mouse models and patients with autoimmune diseases is summarized 89 . To ascertain the extent of autophagy defects, a combination of techniques, such as western blot and flow cytometry, measurement of autophagy makers, fluorescent imaging and electron microscopy, in the presence and absence of lysosomal protease inhibitors, is recommended. Several review articles have described reliable methods dedicated to the measurement of chaperone-mediated autophagy (cmA) activity [228] [229] [230] . Increased expression levels of lAmP2A and HSPA8, two key players in cmA, have been shown to occur in a mouse model of lupus 92 . However, it should be noted that increased expression levels of HSPA8 and lAmP2A starting from a total lysate is only indicative of cmA upregulation; this test is not sufficient to allow any firm conclusion, and it is necessary to examine their expression levels in purified lysosomes or in lysosome-enriched fractions.

NATure revIewS | Drug DiSCOvErY Chaperone modulators. Molecules targeting chaperone proteins involved in lysosomal function have also been designed for potential therapeutic applications. One of these molecules is VER155008, a small molecule inhib itor of HSPA8, a key element of CMA 308, 309 . VER155008 binds to the nucleotide binding domain of HSPA8 and HSP70, and acts as an ATP competitive inhibitor of ATPase and chaperone activity. In a mouse model of AD (5XFAD mice), intraperitoneal treatment with VER155008 reduced the two main pathological fea tures of AD (amyloid plaques and paired helical filament tau accumulation) and improved object recognition, location and episodic like memory 280 . Another molecule, the 21mer phosphopeptide P140 (TABLe 4; Fig. 3 ), was also shown to interact with HSPA8 (Fig. 2) 310 and lodge in the HSPA8 nucleotide binding domain 92, 311 . P140 and VER155008, however, do not have the same mechanism of action, and their effects were not additive 312 . P140 is a phosphorylated analogue of a nominal peptide that was initially spotted in a cellular screening assay using overlapping peptides covering the whole spliceosomal U170K protein and CD4 + T cells collected from the lymph nodes of lupus prone MRL/lpr mice 313 . P140 peptide enters B cells via a clathrin coat dependent endocytosis process to reach early endosomes and then late endosomes/lysosomes 92 . It affects CMA that is hyperactivated in lupus, likely by hampering the CMA mediating chaperone HSPA8 (ReF. 101 ). P140 peptide reduces the excessive expression of HSPA8 and LAMP2A observed in lupus mice, alters the (auto)antigen presentation by MHCII molecules in the MIIC compartment and, consequently, attenuates the activation of autoreactive T cells 92 . A significant diminution of MHC molecule expression at the surface of antigen presenting cells was measured in mice that received the P140 peptide intravenously and on patient's peripheral cells treated ex vivo with the peptide 92,101,314 . As a downstream consequence, the activation of auto reactive B cells and their differentiation into autoantibody secreting cells is repressed 101, 314 . T cells from patients with lupus are no longer responders ex vivo to peptides encompassing CD4 + T cell epitopes 315 . The effect of P140 on CMA was demonstrated in vitro, using a fibroblast cell line that stably expresses a CMA reporter 53, 92 . P140, which selectively targets the CMA/lysosome process and has no effect on mitophagy 316 , has been evaluated in murine models mimicking other rheumatic diseases with very promising results, notably in mice developing SjS features 56 , in mice with neuropsychiatric lupus symp toms 62 and in rats that develop a CIDP like disease with disturbance of both CMA and macroautophagy in sciatic nerves 59 . In clinical trials that included patients with SLE, P140 formulated in mannitol was found to be safe and non immunogenic after several subcutaneous admin istrations of peptide 312, 317, 318 . P140 showed significant efficacy in a multicentre, double blind, phase II trial 317 . This peptide is currently being evaluated in phase III trials in the United States, Europe and Mauritius. In con tinuation, an open label trial including several hundred patients with lupus worldwide is planned.

Another peptide has been discovered that, in con trast to P140, activates CMA 319 . This 24mer peptide called humanin was originally identified from sur viving neurons in patients with AD, and was found to directly enhance CMA activity by increasing substrate binding and translocation into lysosomes. Humanin interacts with HSP90 and stabilizes the binding of this chaperone to CMA cargos as they bind to the lyso somal membrane. These results are important as humanin had been shown to possess some cardioprotective and neuro protective properties in diseases such as AD, cardio vascular disease, stroke, myocardial infarction, diabetes and cancer 320 .

In addition to the targets discussed above, there are a few emerging potential lysosomal therapeutic targets for which there is strong biological validation, but not yet any small molecules in development that target them. An example with likely pharmacological tractability is a lysosomal K + channel called TMEM175, which is impor tant for maintaining the membrane potential and pH stability in lysosomes 321 . Deficiency in TMEM175 may play a critical role in PD pathogenicity 322 . Importantly, the structure of TMEM175 has been recently refined 323 .

Another target for which ligands have not yet been validated is the KCNQ2/3 channel (also named M channel or Kv7.2/7.3 channel). It has been shown in NPC1 disease that reduced cholesterol efflux from lyso somes aberrantly modifies neuronal firing patterns 324 . This disruption of lysosomal cholesterol efflux with decreases in PtdIns(4,5)P2dependent KCNQ2/3 chan nel activity may lead to the aberrant neuronal activity. The cholesterol transporter and PtdIns(4,5)P2 floppase, ABCA1, is responsible for the decline in PtdIns(4,5)P2 that consequently modifies the electrical properties of NPC1 disease neurons. Dysfunction in the activity of KCNQ2/3 or altered levels of PtdIns(4,5)P2, due notably to genetic mutations, might also be involved in other neuropathies (for example, some forms of epilepsy, HD, PD, AD, ALS and Friedrich ataxia). Although further experiments are needed to validate the link discovered between hyperexcitability and cell death in NPC1 disease and other neurodegenerative diseases, small molecules such as retigabine, an anti convulsant drug that keeps KCNQ2/3 channels open, might rep resent important therapeutic tools 324, 325 . Other channel opener ligands of KCNQ2/Q3 include ICA069673 and its derivatives.

Another promising therapeutic target is sphingo myelin phosphodiesterase 1 (SMPD1). Defects in the gene encoding SMPD1 cause Niemann-Pick disease type A and type B. SMPD1 converts sphingomyelin to ceramide, and also has phospholipase C activity. Reduced activity of acid sphingomyelinase, associated with a marked decrease in lysosomal stability, has been described in patients with Niemann-Pick disease, a phenotype that was corrected by treating cells with recombinant HSP70 326 .

Finally, as LAMP2A, a specific lysosomal protein that displays a decisive role in CMA, has been shown to be overexpressed in certain pathological settings such as certain cancers and inflammatory diseases (autoimmune or non autoimmune), downregulating its expression www.nature.com/nrd might be therapeutically beneficial 53, 327 . As mentioned above, however, in other indications there is a defect in LAMP2A. The latter can be due to reduced stabil ity of the CMA receptor and not to decreased de novo synthesis (for example, in ageing) 328 or can result from aggregation to the lysosomal membrane of pathogenic proteins such as α synuclein, ubiquitin carboxy terminal hydrolase L1 (a deubiquitinating enzyme) and mutant tau, known to amass in neurodegenerative disorders (see above). Targeting LAMP2A therefore remains a challenge, although several strategies may be envisaged, for example by controlling de novo synthesis, by ham pering its multimerization into lysosomes (possibly via HSP90 and/or other chaperones) or by regulating the degradation rate of LAMP2A monomers (for reuse) into lysosomes.

Challenges and outlook Current research into lysosomal function and dysfunc tion is revealing novel roles of lysosomes in disease pathogenesis and highlighting new opportunities to treat such lysosomal and autophagy related diseases. As in the case of autophagy modulation 14, 56, 207 , lysosomal activation or inhibition must be investigated with cau tion, as lysosomal activity can be abnormally reduced or enhanced in some organs or tissues and not in others, and, at another scale, lysosome activity can be altered in certain lysosomes and not in others within the same cell. Biodistribution studies in vivo must be undertaken to avoid accumulation of pharmaceuticals in healthy organs or tissues. There is an obvious requirement for safety, to ensure that a drug used as a lysosome modu lator for a particular type of lysosomal disease does not increase vulnerability to another disease.

There is still much to be learned about the intimate working of lysosomes. This is due to the abundance of constitutive elements that comprise these vesicles, the added complexity resulting from their plasticity (ion channels and transporters, acidification and swell ing) and the vast amount of proteins and peptides that are translocated into lysosomes and digested by lytic enzymes. Sensitive analysis methods have allowed important information to be generated about lyso somal membrane proteins, a large majority of which are transporters 8 . However, many questions remain related to how their expression is regulated and how they reg ulate their translocator and chaperoning activities. For example, certain cells only contain so called secretory lysosomes (as in cytotoxic T cells), whereas other cell subsets contain both conventional and secretory lyso somes (as in platelets). Considering the large family of endo lysosomal vesicles, the whole notion of 'secretory' and 'conventional' lysosomes remains a matter of debate. In many instances, lysosomes act as a basal cell metab olism organelle; whereas in other cases, they assist in the regulation of homeostasis through unconventional secretory pathways, known as lysosomal exocytosis, and different signalling mechanisms.

Although several assays used to measure the activ ity of lysosomes have been validated worldwide (Box 2; TABLe 3), they have their limitations, including issues associated with reliability, performance and sensitivity, notably in vivo. Another level of complexity comes from the inherent organelle heterogeneity, which is an issue of tremendous importance. Unfortunately, with the tools and equipment we have in hand today, it is virtually impossible to examine what happens in the lysosomes of an individual patient. The introduction of micro fluidic single cell analysis technologies has enabled cellular populations to be characterized and huge advances to be performed. However, the level of precision has not yet been achieved at the level of lysosomes (0.2-0.5 μm). We know that lysosomes are heterogeneous in nature, composition and activity even in 'normal' settings; they are not all equally competent for autophagy or any other types of activity. Currently, this is obviously the focus of intense research.

Although a certain number of preclinical studies involving lysosomal regulators have been conducted over the years, only a small number of lysosome targeted therapeutics have so far moved into clinical develop ment. One of the biggest advances in developing such strategies would be the identification of a genetic sig nature that would allow those patients most likely to respond to a specific therapy to be selected. However, at this stage of our knowledge of specific lysosome directed drugs and intrinsic lysosomal failures, genetic features that might predict potential responders are still lacking (with the exception of LSDs). Further investi gations are required to achieve this level of knowledge, which obviously will also depend on the type of disease, heterogeneity and frequency.

Another issue associated with the development of lysosometargeted therapeutics relates to delivery. The use of nanovectors represents an attractive delivery method, owing, in particular, to their unique ability to penetrate across cell barriers and, via the endo lysosomal pathway, to preferentially home in on orga nelles such as lysosomes. Several nanoscale galenic forms have been developed to serve as vectors or car riers of proteins, peptides or nucleic acids, and a vast literature describes the many advantages of using such nano structures in nanomedicine. However, safety is a concern as some carbon nanostructures have been claimed to induce nanotoxicity, accompanied by the induction of autophagy and lysosomal dysfunction [329] [330] [331] [332] (reviewed elsewhere [333] [334] [335] [336] .

The purpose of this Review is to gain awareness of the importance of lysosomes in disease, and to encour age the development of novel lysosomal targeted drugs. However, more research is needed to characterize com ponents that are specifically linked to the lysosome, such as LAMP2A and HSPA8, and to more clearly define their specific involvement in lysosome biogenesis and metabolism. Special attention should be given to the mode of administration of lysosome targeted medica tions in order to minimize toxicity and promote specific targeting. It is our hope that a large field of therapeutic applications could emerge from such investigations, encompassing rare and common autoimmune, neuro degenerative and metabolic diseases, as well as cancer, senescence and ageing.

NATure revIewS | Drug DiSCOvErY

Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue

Lysosomal membrane permeabilization and cell death

Signals from the lysosome: a control centre for cellular clearance and energy metabolism

This article is an encyclopaedia of lysosomal physiology

The lysosome as a regulatory hub

Lysosome positioning coordinates mTORC1 activity and autophagy

The coming of age of chaperone-mediated autophagy

Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function

Lysosomal disorders: from storage to cellular damage

Enzyme replacement therapy for lysosomal diseases: lessons from 20 years of experience and remaining challenges

Chemical modulators of autophagy as biological probes and potential therapeutics

Pharmacological regulators of autophagy and their link with modulators of lupus disease

Mechanism and medical implications of mammalian autophagy

Autophagy: a new concept in autoimmunity regulation and a novel therapeutic option

Critical functions of the lysosome in cancer biology

The lysosome as a cellular centre for signalling, metabolism and quality control

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Role of LIMP-2 in the intracellular trafficking of β-glucosidase in different human cellular models

Lysosomal acidification mechanisms

Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology

A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis

MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

Lysosomal storage diseases: from pathophysiology to therapy

This article describes the role of Rab27a, increased phagosome acidification, alteration of subsets of LROs and antigen degradation defects

Melanosomes-dark organelles enlighten endosomal membrane transport

Lysosomes: fusion and function

This review is one of the must-read introductions to the complex and dynamic systems of the lysosomal network

Lysosomerelated organelles: unusual compartments become mainstream

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

Disorders of lysosomerelated organelle biogenesis: clinical and molecular genetics

This article is the first research demonstrating that TFEB controls not only lysosomal biogenesis but also the autophagy network, making TFEB a highly attractive therapeutic target

TFEB and TFE3: linking lysosomes to cellular adaptation to stress

Autophagy in immunity and inflammation

This review brought scientists' attention to the role of autophagy in immunity and inflammation, which prompted intense research into this topic

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB

A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

Macroautophagy substrates are loaded onto MHC class II of medullary thymic epithelial cells for central tolerance

Pathways of antigen processing

Termination of autophagy and reformation of lysosomes regulated by mTOR

Development of research into autophagic lysosome reformation

Unveiling the roles of autophagy in innate and adaptive immunity

The lysosome as a commandand-control center for cellular metabolism

The position of lysosomes within the cell determines their luminal pH

This article highlights the heterogeneity of lysosomes in terms of cell positioning, motility and luminal pH, which all affect lysosomal functionality

Lysosomes and autophagy in cell death control

Lysosomal storage diseases-the horizon expands

Emptying the stores: lysosomal diseases and therapeutic strategies

Alpha-mannosidosis

Treatment for Krabbe's disease: finding the combination

Natural disease history and characterisation of SUMF1 molecular defects in ten unrelated patients with multiple sulfatase deficiency

Abnormal chaperone-mediated autophagy (CMA)

Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease)

A cross-sectional quantitative analysis of the natural history of free sialic acid storage diseasean ultra-orphan multisystemic lysosomal storage disorder

Manipulating autophagic processes in autoimmune diseases: a special focus on modulating chaperone-mediated autophagy, an emerging therapeutic target

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

Lysosomal enzyme activities: new potential markers for Sjögren's syndrome

Rescue of autophagy and lysosome defects in salivary glands of MRL/lpr mice by a therapeutic phosphopeptide

Genetic coding variant in GPR65 alters lysosomal pH and links lysosomal dysfunction with colitis risk

Lysosomes and joint disease

An autophagy-targeting peptide to treat chronic inflammatory demyelinating polyneuropathies

Excess lipid accumulation in cortical neurons in multiple sclerosis may lead to autophagic dysfunction and neurodegeneration

Autophagy dysregulation in ALS: when protein aggregates get out of hand

Autophagy in neuroinflammatory diseases

The lysosome and neurodegenerative diseases

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease

The many faces of autophagy dysfunction in Huntington's disease: from mechanism to therapy

Lysosomal storage diseases

Mitochondrial dysfunction and neurodegeneration in lysosomal storage disorders

Diagnostic challenge for the rare lysosomal storage disease: late infantile GM1 gangliosidosis

Delayed diagnosis of late-onset Pompe disease in patients with myopathies of unknown origin and/or hyperCKemia

Prevalence of Pompe disease in 3,076 patients with hyperCKemia and limb-girdle muscular weakness

The roles of endo-lysosomes in unconventional protein secretion

Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c

A block of autophagy in lysosomal storage disorders

Mucolipidosis type IV: the importance of functional lysosomes for efficient autophagy

Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches

Autophagy in lysosomal storage disorders

The values and limits of an in vitro model of Pompe disease: the best laid schemes o'mice an'men …

The roles of lysosomes in inflammation and autoimmune diseases

Cysteine cathepsins: from structure, function and regulation to new frontiers

Lysosomal cathepsins and their regulation in aging and neurodegeneration

Cathepsin S inhibition combines control of systemic and peripheral pathomechanisms of autoimmune tissue injury

Cathepsin L activity controls adipogenesis and glucose tolerance

Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide

Mediators of inflammation in leukocyte lysosomes: IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes

Defensins-and cathepsin G-ANCA in systemic lupus erythematosus

T cell hyperactivity in lupus as a consequence of hyperstimulatory antigen-presenting cells

Steroids, lyosomes and systemic lupus erythematosus

Autoimmunity to lysosomal enzymes: new clues to vasculitis and glomerulonephritis?

Assessing autophagy in mouse models and patients with systemic autoimmune diseases

Increased expression of cathepsins and obesity-induced proinflammatory cytokines in lacrimal glands of male NOD mouse

Tear cathepsin S as a candidate biomarker for Sjögren's syndrome

This study together with that of Wang et al. (2015) were the first to demonstrate in vivo that regulating CMA and lysosomal dysfunctions could provide potential therapeutic benefit for autoimmune diseases

Systemic lupus erythematosus, complement deficiency, and apoptosis

mTORC2 activity disrupts lysosome acidification in systemic lupus erythematosus by impairing caspase-1 cleavage of Rab39a

Mechanistic target of rapamycin complex 1 expands Th17 and IL-4 + CD4 -CD8 --double-negative T cells and contracts regulatory T cells in systemic lupus erythematosus

Cathepsin K deficiency ameliorates systemic lupus erythematosus-like manifestations in Fas lpr mice

Macroautophagy is deregulated in murine and human lupus T lymphocytes

T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

Blockade of macrophage autophagy ameliorates activated lymphocytes-derived DNA induced murine lupus possibly via inhibition of proinflammatory cytokine production

Autophagy is activated in systemic lupus erythematosus and required for plasmablast development

HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus

Autoimmune diseases: insights from genome-wide association studies

Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus

Genes associated with SLE are targets of recent positive selection

Autophagy in autoimmune disease

Identification of a systemic lupus erythematosus risk locus spanning ATG16L2, FCHSD2, and P2RY2 in Koreans

CD4 T lymphocyte autophagy is upregulated in the salivary glands of primary Sjögren's syndrome patients and correlates with focus score and disease activity

Elevation of autophagy markers in Sjögren syndrome dry eye

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's syndrome

Interferon-γ treatment in vitro elicits some of the changes in cathepsin S and antigen presentation characteristic of lacrimal glands and corneas from the NOD mouse model of Sjögren's syndrome

Cathepsin D agglutinators in rheumatoid arthritis

Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis

Comparison of cathepsins K and S expression within the rheumatoid and osteoarthritic synovium

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin G: the significance in rheumatoid arthritis as a monocyte chemoattractant

Cathepsin S and cathepsin L in serum and synovial fluid in rheumatoid arthritis with and without autoantibodies

Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis

Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role

Passive transfer of demyelination by serum or IgG from chronic inflammatory demyelinating polyneuropathy patients

From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS

Unravelling the pathogenesis of myasthenia gravis

Elevated ATG5 expression in autoimmune demyelination and multiple sclerosis

Neuropsychiatric systemic lupus erythematosus: pathogenesis and biomarkers

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus

Microglia and C9orf72 in neuroinflammation and ALS and frontotemporal dementia

Autophagy and human disease: emerging themes

Disorders of lysosomal acidification-the emerging role of v-ATPase in aging and neurodegenerative disease

Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities

Assessing autophagy in sciatic nerves of a rat model that develops inflammatory autoimmune peripheral neuropathies

Lysosomal proteins as a therapeutic target in neurodegeneration

mTOR as a multifunctional therapeutic target in HIV infection

Autophagy and mitophagy elements are increased in body fluids of multiple sclerosis-affected individuals

Defective autophagy is associated with neuronal injury in a mouse model of multiple sclerosis

Autophagy dysregulation in amyotrophic lateral sclerosis

Autophagy dysregulation in amyotrophic lateral sclerosis

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS

Dysregulation of the autophagy-endolysosomal system in amyotrophic lateral sclerosis and related motor neuron diseases

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models

This pioneering work describes the role of Beclin 1 in the development of amyotrophic lateral sclerosis and highlights the complexity of predicting the effects of manipulating autophagy in a disease context

Exome sequencing uncovers hidden pathways in familial and sporadic ALS

The fragility of cerebral lysosomes in multiple sclerosis

CIDP-the relevance of recent advances in Schwann cell/axonal neurobiology

Molecular mechanisms involved in Schwann cell plasticity

Two faces of Schwann cell dedifferentiation in peripheral neurodegenerative diseases: pro-demyelinating and axon-preservative functions

Studies on the pathogenesis of multiple sclerosis

Autophagy promotes oligodendrocyte survival and function following dysmyelination in a long-lived myelin mutant

Involvement of cathepsin B in the motor neuron degeneration of amyotrophic lateral sclerosis

Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant

Adapting proteostasis for disease intervention

Caspases: a molecular switch node in the crosstalk between autophagy and apoptosis

Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing

Protein aggregation and neurodegenerative disease

Protein aggregation and degradation mechanisms in neurodegenerative diseases

Protein aggregation and neurodegenerative diseases: from theory to therapy

Autophagy: a lysosomal degradation pathway with a central role in health and disease

Endo-lysosomal and autophagic dysfunction: a driving factor in Alzheimer's disease?

Presenilins and γ-secretase in membrane proteostasis

Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives

Drug repositioning for Alzheimer's disease

Processing of the beta-amyloid precursor. Multiple proteases generate and degrade potentially amyloidogenic fragments

Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease

Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: evidence for early up-regulation of the endosomallysosomal system

Novel cathepsin D inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus

Does intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein trigger early neurotoxicity in Alzheimer's disease?

Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations

The complicated relationship between Gaucher disease and parkinsonism: insights from a rare disease

Lysosomal impairment in Parkinson's disease

& Rubinsztein, D. C. α-Synuclein is degraded by both autophagy and the proteasome

This article discovered that α-synuclein, a protein associated with several neurodegenerative diseases

Abberant α-synuclein confers toxicity to neurons in part through inhibition of chaperonemediated autophagy

Lysosomal degradation of alphasynuclein in vivo

Cathepsin D is the main lysosomal enzyme involved in the degradation of α-synuclein and generation of its carboxy-terminally truncated species

The role of cathepsin D in the pathogenesis of human neurodegenerative disorders

Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo

Therapeutic approaches to Huntington disease: from the bench to the clinic

Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus

Age-related changes in activities and localizations of cathepsins D, E, B, and L in the rat brain tissues

Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

Autophagy regulates the processing of amino terminal huntingtin fragments

Lysosomal proteases are involved in generation of N-terminal huntingtin fragments

Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin

Dysregulation of core components of SCF complex in poly-glutamine disorders

Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease

Role of chaperone-mediated autophagy in degrading Huntington's diseaseassociated huntingtin protein

A photoconvertible fluorescent reporter to track chaperone-mediated autophagy

The role of chaperone-mediated autophagy in huntingtin degradation

Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein

Mutant Huntingtin is secreted via a late endosomal/lysosomal unconventional secretory pathway

Altered lysosomal positioning affects lysosomal functions in a cellular model of Huntington's disease

Lysosomal dysfunction in neurodegeneration: the role of ATP13A2/PARK9

Protein misfolding in neurodegenerative diseases: implications and strategies

The autophagy-lysosomal pathway in neurodegeneration: a TFEB perspective

Overexpression of TFEB drives a pleiotropic neurotrophic effect and prevents Parkinson's disease-related neurodegeneration

TFEB dysregulation as a driver of autophagy dysfunction in neurodegenerative disease: molecular mechanisms, cellular processes, and emerging therapeutic opportunities

Transcription factor EB Is selectively reduced in the nuclear fractions of Alzheimer's and amyotrophic lateral sclerosis brains

Pharmacologic agents targeting autophagy

Autophagy in the renewal, differentiation and homeostasis of immune cells

Enzyme replacement for lysosomal diseases

Enzyme replacement therapy: lessons learned and emerging questions

Enzyme replacement therapies: what is the best option?

Early higher dosage of alglucosidase alpha in classic Pompe disease

A multicenter, open-label, phase III study of Abcertin in Gaucher disease

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection

Autophagy modulation as a potential therapeutic target for diverse diseases

The therapeutic and pathogenic role of autophagy in autoimmune diseases

Pharmacological autophagy regulators as therapeutic agents for inflammatory bowel diseases

Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells

Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation

Autophagy and rheumatoid arthritis: current knowledges and future perspectives

Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines

Evidence for risk of cardiomyopathy with hydroxychloroquine

Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency

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Pharmacological enhancement of α-glucosidase by the allosteric chaperone Nacetylcysteine

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The pharmacological chaperone 1-deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid alpha-glucosidase

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A bicyclic 1-deoxygalactonojirimycin derivative as a novel pharmacological chaperone for GM1 gangliosidosis

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Dendritic cells treated with chloroquine modulate experimental autoimmune encephalomyelitis

Hydroxychloroquine for autoimmune diseases

Sirolimus in patients with clinically active systemic lupus erythematosus resistant to, or intolerant of, conventional medications: a single-arm, open-label, phase 1/2 trial

Modulation of the immune response in rheumatoid arthritis with strategically released rapamycin

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Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity

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Discovery of a small-molecule probe for V-ATPase function

Small molecule activators of TRPML3

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VER-155008, a small molecule inhibitor of HSP70 with potent anticancer activity on lung cancer cell lines

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Humanin is an endogenous activator of chaperone-mediated autophagy

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Treatments for lysosomal storage disorders

Treatment of Gaucher disease with an enzyme inhibitor

Miglustat in Niemann-Pick disease type C patients: a review

Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study

Parkinson's disease prevalence in Fabry disease: a survey study

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Pharmaceutical chaperones and proteostasis regulators in the therapy of lysosomal storage disorders: current perspective and future promises

Treatment of lysosomal storage diseases: recent patents and future strategies

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Treatment strategies for lysosomal storage disorders

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Cathepsin S as an inhibitor of cardiovascular inflammation and calcification in chronic kidney disease

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Bafilomycin A1 inhibits chloroquineinduced death of cerebellar granule neurons

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons

Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-ATPases

The curious case of vacuolar ATPase: regulation of signaling pathways

The hidden potential of lysosomal ion channels: a new era of oncogenes

TRPML1: the Ca (2+) retaker of the lysosome

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

Regulation of TRPML1 function

Differential mechanisms of action of the mucolipin synthetic agonist, ML-SA1, on insect TRPML and mammalian TRPML1

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Identification of apilimod as a firstin-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells

Functional analysis of Hsp70 inhibitors

The spliceosomal phosphopeptide P140 controls the lupus disease by interacting with the HSC70 protein and via a mechanism mediated by γδ T cells

Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival

Resetting the autoreactive immune system with a therapeutic peptide in lupus

T cell recognition and therapeutic effect of a phosphorylated synthetic peptide of the 70K snRNP protein administered in MRL/lpr mice

Lupus regulator peptide P140 represses B cell differentiation by reducing HLA class II molecule overexpression

Selective modulation of CD4 + T cells from lupus patients by a promiscuous, protective peptide analog

The mitochondrion-lysosome axis in adaptive and innate immunity: effect of lupus regulator peptide P140 on mitochondria autophagy and NETosis

P140 peptide in patients with systemic lupus erythematosus: a randomised, doubleblind, placebo-controlled phase IIb clinical trial

The importance of implementing proper selection of excipients in lupus clinical trials

Humanin is an endogenous activator of chaperone-mediated autophagy

Central effects of humanin on hepatic triglyceride secretion

TMEM175 is an organelle K + channel regulating lysosomal function

TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

Niemann-Pick type C disease reveals a link between lysosomal cholesterol and PtdIns(4,5)P2 that regulates neuronal excitability

Functional upregulation of the M-current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons

Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

Chaperone-mediated autophagy is required for tumor growth

Restoration of chaperonemediated autophagy in aging liver improves cellular maintenance and hepatic function

Role for nanomaterialautophagy interaction in neurodegenerative disease

A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt-TSC2-mTOR signaling

Tuning cell autophagy by diversifying carbon nanotube surface chemistry

Silica nanoparticles enhance autophagic activity, disturb endothelial cell homeostasis and impair angiogenesis

Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

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Berberine as a potential autophagy modulator

The authors apologize to all those whose work is not cited due to space limitations. They gratefully acknowledge Hélène Jeltsch-David for critically reading the manuscript. This research was funded by the French Centre National de la Recherche Scientifique, Région Alsace, the Laboratory of Excellence Medalis (ANR-10-LABX-0034), Initiative of Excellence (IdEx), Strasbourg University, and ImmuPharma France. S.M. is grateful to the University of Strasbourg Institute for Advanced Study (USIAS) for funding F.W., and acknowledges the support of the TRANSAUTOPHAGY COST Action (CA15138), the Club francophone de l'autophagie (CFATG) and the European Regional Development Fund of the European Union in the framework of the INTERREG V Upper Rhine programme.

All authors made substantial, direct and intellectual contribution to the work and approved it for publication.

S.M. discloses the following conflicts of interest: research funding (paid to institution) and a past consultant for ImmuPharma; co-inventor of CNRS-ImmuPharma patents on P140 peptide; owns ImmuPharma shares. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S.R.B. and F.W. declare no competing interests.

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