COMMENTARY ARTICLE SERIES: CELL BIOLOGY AND DISEASE

Danon disease – dysregulation of autophagy in a multisystemdisorder with cardiomyopathyTeisha J. Rowland, Mary E. Sweet, Luisa Mestroni and Matthew R. G. Taylor*

ABSTRACTDanon disease is a rare, severe X-linked form of cardiomyopathycaused by deficiency of lysosome-associated membrane protein 2(LAMP-2). Other clinical manifestations include skeletal myopathy,cognitive defects andvisual problems.Although individualswithDanondisease have been clinically described since the early 1980s, theunderlying molecular mechanisms involved in pathologicalprogression remain poorly understood. LAMP-2 is known to beinvolved in autophagy, and a characteristic accumulation ofautophagic vacuoles in the affected tissues further supports the ideathat autophagy is disrupted in this disease. The LAMP2 gene isalternatively spliced to form three splice isoforms, which are thought toplay different autophagy-related cellular roles. This Commentaryexplores findings from genetic, histological, functional and tissueexpression studies that suggest that the specific loss of the LAMP-2Bisoform, which is likely to be involved in macroautophagy, plays acrucial role in causing theDanonphenotype.Wealso compare findingsfrom mouse and cellular models, which have allowed for furthermolecularcharacterizationbut havealsoshownphenotypic differencesthat warrant attention. Overall, there is a need to better functionallycharacterize the LAMP-2B isoform in order to rationally explore moreeffective therapeutic options for individuals with Danon disease.

KEY WORDS: Danon disease, Autophagy, Lysosome-associatedmembrane protein 2, LAMP-2, Cardiomyopathy, Medical genetics,Macroautophagy

IntroductionDanon disease is a rare but severe X-linked form of cardiomyopathythat usually leads to profound hypertrophic cardiomyopathy (HCM;a thickening of the heart muscle) and causes death or a need forcardiac transplantation in males by the third decade (Fig. 1A)(D’souza et al., 2014; Taylor et al., 2007).When Danon diseasewasfirst described in 1981, ultrastructural analysis of muscle biopsiesfrom two boys with the disease showed that their cells containedabundant glycogen particles, primarily within lysosomal vacuoles(Danon et al., 1981). Because of these observations, the diseasewasoriginally postulated to be due to a glycogen storage defect.However, although glycogen-containing autophagic vacuoles havecontinued to be reported in affected individuals (Cheng and Fang,2012), genetic, histological and ultrastructural analyses haverevealed that disruption of autophagy – a process by which cellsdisassemble dysfunctional or unneeded molecular and cellularcomponents for reuse – is the probable underlying mechanism ofDanon disease.Danon disease is caused by mutations in the lysosome-associated

membrane protein 2 (LAMP2) gene, usually leading to deficiency of

the LAMP-2 protein (Eskelinen, 2005; Nishino et al., 2000).LAMP-2 is an abundant constituent of the membrane of thelysosome, which is a eukaryotic organelle containing digestiveenzymes that break down its contents (Konecki et al., 1994).Unsurprisingly, based on its location, LAMP-2 is involved inautophagy, with its three different splice isoforms (LAMP-2A,LAMP-2B, and LAMP-2C) each playing different autophagy-related roles. There are three main types of autophagy:macroautophagy, chaperone-mediated autophagy (CMA) andmicroautophagy (Mrschtik and Ryan, 2015). The LAMP-2Aisoform has been clearly shown to be involved in CMA(Eskelinen et al., 2005; Schneider and Cuervo, 2014), whereas therole of LAMP-2B has been characterized to a much lesser extent,but it is likely to be involved in macroautophagy (Bandyopadhyayet al., 2008; Tanaka et al., 2000; Nishino et al., 2000).Macroautophagy is the most prevalent form of autophagy and isthought to be the type of autophagy that is most affected in Danondisease (Endo et al., 2015; Tanaka et al., 2000).

In this Commentary, following a brief summary of clinicalmanifestations, the molecular and cellular aspects of Danon diseasewill be presented; these findings suggest that loss of the LAMP-2Bisoform has a crucial role in causing Danon disease throughmediating the disruption of macroautophagy and potentially othervesicle-trafficking processes. We will then discuss thecharacterizations conducted using mouse models separately owingto the phenotypic differences observed when these models arecompared to the human disease. Based on these findings, we arguethat a better understanding of the functions of the LAMP-2Bisoform is needed before more effective therapeutics for Danondisease individuals can be developed.

Clinical manifestations of Danon diseaseMales with Danon disease first show symptoms at 12 years of age,on average, and the majority (88%) develop HCM. By the time theyare approximately 18 years old, the disease has led to cardiactransplantation or death (Boucek et al., 2011). Severe HCM,arrhythmias and a need for cardiac transplantation are the mostmorbid features. Although it is an X-linked disease, heterozygousfemales (i.e. carriers) are also affected but develop a more dilatedcardiac phenotype and a progression of disease that occursapproximately 15 years later than in male counterparts (Prallet al., 2006; Boucek et al., 2011). Postmortem and explantedhearts display substantial fibrosis (D’souza et al., 2014) (Fig. 1B),and many affected individuals (males and females) developarrhythmias that lead to sudden cardiac death. Specifically,ventricular (Hedberg Oldfors et al., 2015; Miani et al., 2012;Maron et al., 2009; Lobrinus et al., 2005) and atrial (Charron et al.,2004) arrhythmias have both been reported and are often severe,even in women (Miani et al., 2012). Additionally, individuals withDanon disease can also develop (usually relatively mild) skeletalmyopathy (80–90% of males), primarily mild cognitive defects and

Cardiovascular Institute and Adult Medical Genetics Program, University ofColorado Denver, Aurora, CO 80045, USA.

*Author for correspondence (matthew.taylor@ucdenver.edu)

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learning disabilities (70–100% of males), and visual problems (69%of males) (Thiadens et al., 2012; Schorderet et al., 2007; Prall et al.,2006; D’souza et al., 2014). For a complete and recent review ofclinical descriptions of affected individuals, see D’souza et al.(2014). For reviews discussing other diseases that share clinicalsimilarities with Danon disease, including Pompe disease,Niemann-Pick type C and other lysosomal storage diseases, seeRuivo et al. (2009), D’souza et al. (2014) and Pastores and Hughes(2015). Although Danon disease shares clinical manifestations withother diseases, the primary distinguishing feature of Danon diseaseis a probable disruption of autophagy, as evidenced by themolecular phenotype, which will be explored next.

Molecular phenotype of Danon diseaseSince its original description in 1981, subsequent findings haverevealed that disrupted autophagy is the probable underlyingmechanism of Danon disease, caused by mutations in the LAMP2gene that usually lead to a loss of expression of LAMP-2. Given theroles of LAMP-2 in autophagy – which will be discussed in detaillater – disruption of autophagy is not surprising. Accordingly, incardiac tissue of individuals with Danon disease, dramatic increasesin cytoplasmic vacuolation are apparent (Endo et al., 2015; He et al.,2014) (Fig. 1C). In these cells, expression of microtubule-associatedprotein 1 light chain 3 proteins (encoded by MAP1LC3A,MAP1LC3B, MAP1LC3C; collectively referred to here as LC3),markers of autophagy that are discussed more later, as well asubiquitin, which comprises the ubiquitin–proteasome system, areincreased in myocardium autophagic vacuoles, suggesting that thematuration and clearance of autophagosomes is stunted (Endo et al.,2015). The number of lipid droplets in these cells is also increased.In addition, the diseased cardiomyocytes are hypertrophic, and themyocardium undergoes degeneration, as evidenced by lipofuscinaccumulation, which can be indicative of damage to cellularorganelles, including lysosomes and disarray of myofibrils (Heet al., 2014; Endo et al., 2015).Recently, fibroblast biopsies taken from two males with Danon

disease were reprogrammed into induced pluripotent stem cells(iPSCs) and differentiated into cardiomyocytes (iPSC-CMs); thishas allowed for further phenotypic and functional studies of thedisease on a controlled molecular level (Hashem et al., 2015). Thesederived cardiomyocytes displayed molecular and cellularphenotypes that were similar to those observed in Danon diseasetissues and included accumulation of intracytoplasmic vacuoles,hypertrophy and impaired autophagic flux with a nearly completedeficiency of mature autophagic vacuoles, as assessed using an LC3reporter (discussed further in the Autophagy section) (Hashemet al., 2015). Interestingly, both skin fibroblasts and iPSC-CMs fromthe Danon disease individuals had significant accumulations of

early autophagic vesicles (compared to wild-type cells), andstarvation (which induces autophagy) increased the number ofsuch vesicles in the iPSC-CMs, but not in the fibroblasts; this mightbe related to the metabolically active nature of cardiac cells. LongerCa2+-decay times were also observed in the Danon iPSC-CMs,which is consistent with a decline in systolic and diastolic function(seen in heart failure), and in other iPSC-CMs used to model heartfailure phenotypes. Furthermore, the iPSC-CMs displayedmitochondrial defects, including increased levels of mitochondrialoxidative stress, and increased levels of apoptosis (Hashem et al.,2015). These effects could potentially be related becausemitochondria normally produce reactive oxygen species (ROS) asoxidative phosphorylation by-products; therefore, dysfunctionalmitochondria can produce excessive amounts of ROS and might bemore prone to activate apoptosis (Shires and Gustafsson, 2015).

Histological examinations of skeletal muscle biopsies fromDanon individuals, which have been more frequently reportedowing to the relative ease of obtaining these tissues, have resulted insimilar findings and are also indicative of autophagy disruption. Forinstance, in skeletal muscle biopsies, an accumulation of vacuolarstructures are consistently found; these contain acetylcholineesterase, proteins of the sarcolemma (the muscle fiber cellmembrane) and extracellular matrix proteins (typically those ofthe basal lamina, such as dystrophin, laminin and spectrin) (Douguet al., 2009; Endo et al., 2015; Yang and Vatta, 2007), and areknown as autophagic vacuoles with unique sarcolemmal features(AVSFs). AVSFs should be located near the peripheral sarcolemmaif they are sarcolemma-derived but, because they are instead foundscattered throughout the cytoplasm, it is thought that these AVSFsare generated through a poorly understood de novo biogenesisprocess (Endo et al., 2015). Disease-associated skeletal musclevacuoles have also been reported to accumulate glycogen (Bertiniet al., 2005; He et al., 2014; Yang and Vatta, 2007), degeneratingmitochondria (Yang and Vatta, 2007), lipids (Bertini et al., 2005)and basophilic granules (implicating buildup of lysosomalorganelles in the myofibers) (He et al., 2014; Sugie et al., 2005;Endo et al., 2015; Dougu et al., 2009), and to express increasedlevels of LC3 and ubiquitin (Endo et al., 2015).

Pathological studies of brain tissue from individuals with Danondisease have been limited, although an autopsy study on oneindividual with intellectual disability has been reported (Furutaet al., 2013). Here, histological andmicroscopic analysis of the braintissue revealed similar findings to those reported for skeletal andcardiac muscle biopsies, namely the striking presence of lipofuscin-like granules containing glycogen, as well as lysosomal storageabnormalities as revealed by electron microscopy analyses and,moreover, a lack of normal autophagic vacuoles (Furuta et al., 2013;Endo et al., 2015). However, unlike the observations made in

Fig. 1. Explanted cardiac muscle from a 14-year-old male with Danon disease. Images show (A) a cross-section of explanted heart with biventricularhypertrophy and fibrosis, (B) a photomicrograph (with trichrome stain) of cardiomyocytes (red) and extensive fibrosis (blue) and (C) a photomicrograph (withhematoxylin and eosin stain) revealing extensive vacuolization. Scale bars: 100 μm. Reprinted from Taylor et al. (2007) with permission. Copyright 2007, NaturePublishing Group.

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skeletal and cardiac muscle biopsies, the studied neurons did notappear to express LC3 and exhibited only low levels of ubiquitin.Clearly, further investigation is needed to better understand thepathological phenotype in the brain.Some clinical studies have described the ophthalmic

manifestations of individuals with Danon disease (Thiadens et al.,2012; Prall et al., 2006; Lobrinus et al., 2005). Both maleindividuals with Danon disease and female carriers have beenfound to have retinopathy, including peripheral pigmentaryretinopathy (Prall et al., 2006) and peripheral ‘salt-and-pepper’retinopathy (Schorderet et al., 2007; Thiadens et al., 2012), althoughsometimes the condition is milder in females. In one study, bothenrolled males had a near-complete loss of pigmentation of theretinal pigment epithelium (RPE) in the macula (Prall et al., 2006),whereas a boy from another study had RPE hypopigmentation(Schorderet et al., 2007). Another study of a family found that twoout of four male members with Danon disease developed cone-roddystrophy, with thinning of the RPE and overlying photoreceptorlayers (Thiadens et al., 2012). Photoreceptor cells (comprised ofrods and cones) absorb photons of light through components calledphotoreceptor outer segments (POSs). Each day, at least 2000–4000POSs are shed by photoreceptors and broken down by theunderlying RPE layer through phagocytosis (Thiadens et al.,2012). Unlike autophagy, phagocytosis requires import of itscargo, but similar to autophagy, its cargo must be degraded throughlocalization to, and fusion with, the lysosome (Nandrot, 2014).Some key phagocytic factors have been identified, includingmyosin VIIA – a mechanochemical protein thought to transportnascent POS-based vesicles (i.e. phagosomes) into the cell (Gibbset al., 2003) – as well as proteins and proteases (e.g. βA3-crystallin,A1-crystallin and cathepsin D) that are involved in lysosomaldegradation following fusion of the phagosome with the lysosome(Nandrot, 2014; Valapala et al., 2016). However, it is likely thatother factors with an active role in retinal phagocytosis are yet to beidentified, particularly because these molecular mechanisms haveonly been characterized using nocturnal rodent models, andphagocytotic cycles are different in diurnal animals, such ashumans (Nandrot, 2014). Interestingly, disruption of retinalphagocytosis leads to lipofuscin accumulation (Nandrot, 2014),which is also seen in Danon individuals, as described above.LAMP-2 is highly expressed in RPE lysosomes (Schorderet et al.,2007), indicative of a role in the retinal phagocytotic process.Indeed, mutation of the LAMP2 gene could ultimately lead to theloss of RPE and photoreceptors, and thus to a loss of visual acuity(Thiadens et al., 2012).

LAMP-2 structure and isoformsUnderstanding the structure of LAMP-2 and its splice isoforms isimportant for determining their varying roles in autophagy, and howthese might be disrupted in Danon disease. The human LAMP-2protein contains nine exons, with exons 1 to 8 and part of exon 9making up the lysosome lumenal domain. In this large lumenaldomain, four loops are predicted to form through disulfide bridgesand flank a proline-rich ‘hinge’ region (Eskelinen et al., 2005). Thehinge region acts as a linker for two similar glycosylated domains,each of which contains two of the disulfide bridges (Wilke et al.,2012). The lumenal domain of LAMP-2 is heavily glycosylated toavoid degradation due to the low pH of the lysosome (Mrschtik andRyan, 2015). For additional details on the recently solved crystalstructure of the LAMP-2 protein – with a focus on the lumenaldomain – see Wilke et al. (2012). The remaining part of exon 9comprises both a single transmembrane domain (24 amino acids

long) and a short cytoplasmic tail (11 amino acids long) at the C-terminal end (Eskelinen et al., 2005; Konecki et al., 1994, 1995).The cytoplasmic tail is likely to function as a receptor for the uptakeof proteins and, occasionally, other molecules (such as RNA andDNA) into the lysosome for their degradation (D’souza et al., 2014;Nishino et al., 2000). It is worth noting that although LAMP-1,which belongs to the same family as LAMP-2, shares 37% aminoacid sequence homology with LAMP-2, these proteins have beenfound to be distinct and to have largely separate functions(Eskelinen, 2006). Overall, the LAMP1-knockout mousephenotype is relatively mild (Eskelinen, 2006), and although theremight be shared functions between LAMP-1 and LAMP-2, LAMP-1 levels have not been found to increase to compensate for blockageof CMA, which is the best characterized function of LAMP-2(Massey et al., 2006), nor does transient expression of LAMP-1alleviate cholesterol accumulation (Schneede et al., 2011). For adetailed comparison of LAMP-1 and LAMP-2 functions, seeEskelinen (2006).

As mentioned previously, the LAMP2 gene can be alternativelyspliced to give rise to three splice isoforms, LAMP-2A, LAMP-2Band LAMP-2C. For a thorough review of the isoform nomenclatureand standardization across different species, see Eskelinen et al.(2005). Exon 9 of LAMP-2 pre-messenger RNA (mRNA) isalternatively spliced to create the three splice isoforms, which areidentical in their lysosome lumenal domains but differ in thetransmembrane domain and cytoplasmic tail (Fig. 2C). LAMP-2Aplays a role in CMA, whereas the other isoforms do not appear to beinvolved in CMA (Eskelinen et al., 2005). Studies suggest thatLAMP-2B could instead be involved in macroautophagy, primarilybased on the impaired fusion seen in individuals with mutations inLAMP-2B only (Bandyopadhyay et al., 2008; Tanaka et al., 2000;Nishino et al., 2000). Both CMA and macroautophagy will bediscussed in detail later. LAMP-2C is less characterized but isthought to be primarily, and potentially solely, involved in thedegradation of RNA and DNA (RNautophagy and DNautophagy,respectively) based on pull-down assays identifying interactingproteins as being RNA- or DNA-binding proteins (Fujiwara et al.,2013, 2014). Owing to these differences in function and tissueexpression, which will be explored more in the following sections,LAMP-2A and LAMP-2B are thought to be more relevant to Danondisease and, consequently, these isoforms will receive moreattention than LAMP-2C in this Commentary.

In their cytoplasmic tails, LAMP-2A and LAMP-2B share 27%(3 out of 11) identical amino acids, and an additional 27% (3 out of11) of their amino acid residues are similar, with 45% (5 out of 11)of residues not showing any similarity. Such differences in thecytoplasmic tail might enable the isoforms to perform separatereceptor-mediated functions (i.e. CMA and macroautophagy) andmake this region, possibly, of most relevance to Danon disease(D’souza et al., 2014; Nishino et al., 2000).

Role of LAMP-2B in Danon diseaseAlthough most LAMP2 mutations leading to Danon disease arepredicted to result in deficiency of all three of the LAMP-2isoforms, isoform-specific Danon disease mutations have only sofar been found in the LAMP-2B isoform. This suggests that LAMP-2B deficiency is potentially sufficient (owing to the reporting ofisoform-specific mutations) and necessary (all mutations affectLAMP-2B) to cause Danon disease (D’souza et al., 2014; Bouceket al., 2011; Nishino et al., 2000) (Table 1). For extensive lists ofreported LAMP-2 mutations in individuals with Danon disease, seeCheng and Fang (2012), Dougu et al. (2009) and D’souza et al.,

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(2014). Accordingly, deficiency of LAMP-2B alone has been foundto cause ‘the full Danon syndrome’, as stated by the authors,including HCM, skeletal myopathy and the cognitive defects thathave been detected in one individual (Nishino et al., 2000). In one ofthe individuals that we have studied, LAMP-2B deficiency was alsofound to result in a typical case of Danon disease, requiring cardiactransplantation (Boucek et al., 2011). However, in another

individual, LAMP-2B deficiency alone was found to only cause amilder presentation Danon syndrome, with mild left ventriculardiastolic dysfunction, limb-girdle muscle weakness and sub-clinicalneuropathy (Hong et al., 2012). It is also worth noting that a LAMP-2B missense mutation that results in apparently normal levels (butirregular distribution) of LAMP-2B expression in skeletal musclebiopsies from multiple members of one family has been associated

KeyIdentical residuesSimilar residuesCysteine residues in disulfide bridges

Fig. 2. Amino acid sequence alignments of the LAMP-2A and LAMP-2B isoforms in human and mouse. Alignments show (A) human and mouse LAMP-2(exons 1–8), (B) human and mouse LAMP-2A and LAMP-2B isoforms (exon 9 only), which are the LAMP-2 isoforms most relevant to Danon disease, and(C) human LAMP-2A and LAMP-2B isoforms (exon 9 only). Blue and yellow backgrounds indicate identical and similar amino acids, respectively. Graybackgrounds indicate cysteine residues thought to be involved in disulfide bridges. Black, red and green font colors indicate lumenal, transmembrane andcytoplasmic residues, respectively (Eskelinen et al., 2005). Amino acid sequences were obtained from Ensembl, and alignments were generated using theBioinformatics Organization Multiple Align Show tool.

Table 1. Evidence for the involvement of the LAMP-2A and LAMP-2B isoforms in the development of Danon disease

Characteristics

General findings inindividuals with Danondisease

Findings related to theLAMP-2A isoform

Findings related to the LAMP-2Bisoform References

Genotype Usually deficient in allthree LAMP-2 isoforms

Deficiency of this isoformalone has not beenreported in Danondisease individuals

Deficiency of this isoform alonehas been associated with fullDanon syndrome or a milderphenotype

Missense mutation associatedwith mild cardiac phenotype andsevere skeletal myopathy

D’souza et al., 2014; Boucek et al.,2011; Nishino et al., 2000; Honget al., 2012; van der Kooi et al.,2008

Protein expressionlevels in affectedtissues

Cardiac, skeletal muscle,retinal tissue andcognitive functionsmost impaired

Low levels in skeletalmuscle and brain tissue

Similar levels to LAMP-2Bin the heart

High levels in unaffectedtissues: placenta, lung,liver, kidney, pancreas,prostate

High levels in skeletal muscle andbrain tissue

Similar levels to LAMP-2A in theheart

Konecki et al., 1995; Furuta et al.,1999

Autophagyassociation

Accumulation ofautophagic vesicles

Plays a role in CMA Not involved in CMAThought to be involved inmacroautophagy

Tanaka et al., 2000; Nishino et al.,2000; Eskelinen et al., 2005;Bandyopadhyay et al., 2008;Endo et al., 2015

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with different Danon-disease-like symptoms, including apotentially much milder cardiac phenotype (although there wassudden cardiac death at the age of 28 of one female family member),potentially more severe and progressive muscle weakness (which isuncommon in Danon disease individuals), decreased visual acuityand normal intellectual abilities (van der Kooi et al., 2008).Interestingly, upon ultrastructural examination, accumulation ofautophagic vacuoles containing glycogen, lipid droplets andlipofuscin were also observed in this family (van der Kooi et al.,2008). It is possible that compared to loss of expression of all threeisoforms, LAMP-2B deficiency or dysfunction alone mighttypically result in milder Danon-disease-like cardiac and cognitivephenotypes but still give rise to similar or more severe skeletalmyopathy. This could be explained by the tissue-specific expressionlevels of the normal LAMP-2 isoforms, their roles in these tissues,and/or cross-talk and compensatory mechanisms occurring betweenthe different forms of autophagy (Kaushik et al., 2008).Supporting the idea of a key role of LAMP-2B in the causality of

Danon disease is the tissue-specific expression of the differentLAMP-2 isoforms; LAMP-2B appears to be more highly expressedin the most affected tissues compared to LAMP-2A and LAMP-2Cunder normal conditions (Table 1). Human LAMP-2B hasspecifically been found to be more abundantly expressed at themRNA level in skeletal muscle and brain tissue than the LAMP-2Aisoform, which is present at only low levels in these tissues(Konecki et al., 1995; Furuta et al., 1999; Rothaug et al., 2015).LAMP-2B is most highly expressed in skeletal muscle (Koneckiet al., 1995), which could be the reason why the most strikingdisease symptom reported in members of a family with a LAMP-2Bmissense mutation is severe and progressive skeletal muscleweakness (van der Kooi et al., 2008). Human LAMP-2A, bycontrast, is more ubiquitously expressed; it is expressed in placenta,lung, liver, kidney, pancreas and prostate tissues (Konecki et al.,1995; Furuta et al., 1999). Both LAMP-2A and LAMP-2B arethought to be expressed at similar levels in the human heart,although this is based on limited studies (Konecki et al., 1995; Yangand Vatta, 2007). Investigations in human tissues have not beenreported; however, in 2013, a tissue expression study of LAMP-2CmRNA in mice found this isoform to be most highly expressed inbrain tissue and present at lower levels in other tissues, includingdetectable levels in skeletal muscle and the heart (Fujiwara et al.,

2013), although a later study detected no LAMP-2C mRNA in themouse brain (Rothaug et al., 2015). In agreement with some of thesefindings, we have conducted RNA sequencing (RNA-Seq) analysisof normal human iPSC-CMs and normal explanted human hearts,and found that expression levels of the LAMP-2A and LAMP-2Bisoforms are similar in these cells and tissues, with both isoformssignificantly more highly expressed than LAMP-2C (unpublisheddata; T.J.R., M.E.S., L.M., M.R.G.T., Eric Adler, Sherin Hashemand Carmen Sucharov). It is possible that the relative expressionlevels of the isoforms are altered depending on certain metabolicconditions, such as starvation (which induces autophagy); this couldaffect the relevance of the different isoforms to the Danon diseasephenotype. Additional expression studies have been conducted inmice, and these will be explored separately later owing tophenotypic differences observed between individuals with Danondisease and mouse models. The molecular phenotype of individualswith Danon disease described above, wherein macroautophagyspecifically appears to be impacted, is also supportive of a key rolefor LAMP-2B (Table 1).

AutophagyMacroautophagy is thought to be the type of autophagy mostaffected in Danon disease, owing to the accumulation of autophagicvacuoles in muscle biopsies and the idea that the disease is largelycaused by loss of expression of the LAMP-2B isoform, which isthought to be involved in macroautophagy (Table 1) (Endo et al.,2015; Tanaka et al., 2000). In macroautophagy, cargo is engulfed bydouble-membraned vesicles called autophagosomes, which fusewith the lysosome to degrade their contents; this typically results infree fatty acids and amino acids for the cell to reuse (Nishida et al.,2015; Mrschtik and Ryan, 2015; Massey et al., 2008) (Fig. 3).Macroautophagy can be selective or non-selective. CMA, bycontrast, is receptor mediated and generally a more selective processwhere vesicles are not needed for shuttling cargo to the lysosome(Schneider and Cuervo, 2014). The cytoplasmic tail of LAMP-2A isthought to function as a receptor in CMA (Endo et al., 2015). In therecently discovered degradation processes of RNautophagy andDNautophagy, which are mediated by LAMP-2C, this isoform actsas a receptor by interacting with RNA- or DNA-binding proteins,respectively, to allow RNA or DNA to be directly taken up intolysosomes (Fujiwara et al., 2013, 2014). Because nucleic acids have

Isolationmembrane

(phagophore) Autophagosome

Endosome

Amphisome

Autolysosome

Lysosome

Chaperone–substratecomplex

Macroautophagy Chaperone-mediatedautophagy

MitochondrionRibosome Peroxisome

Membrane GlycogenLipid granules Protein

Hsc70LAMP-2BLAMP-2AKey

Fig. 3. Mammalian pathways of macroautophagy and chaperone-mediated autophagy (CMA). In macroautophagy, a variety of cargo can be engulfed by anautophagosome, which fuses with a lysosome to form an autolysosome to then degrade its contents. Alternatively, an autophagosome can fuse with anendosome to form an amphisome, which then fuses with the lysosome. The processes of autophagosome and amphisome fusion with the lysosome are thoughtto require LAMP-2B, although any direct binding partners of LAMP-2B, which are important for these processes, are unknown. In CMA, target proteins arerecognized and bound by heat shock cognate protein 70 (Hsc70; also known as HSPA8), which forms a chaperone–substrate complex that is transported to thelysosome, where interaction with LAMP-2A transport the target protein across the lysosome membrane.

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not been reported in the autophagic vacuoles that are characteristicof Danon disease, these degradation processes will not be discussed;please see Fujiwara et al. (2013, 2014) for details. Macroautophagyand CMA are explored in more detail below with a focus onmacroautophagy because it appears to be the most affected in Danondisease (Endo et al., 2015; Tanaka et al., 2000).In macroautophagy, initially a small flat membrane sack of

cisterna, called an isolation membrane (or phagophore), forms in thecytoplasm (Hamasaki et al., 2013; Nishida et al., 2015). Severalautophagy-related (Atg) proteins are involved in forming, expandingand elongating the isolation membrane. (For in-depth discussions ofthese Atg proteins, see Nemchenko et al., 2011; Mizushima et al.,2011; Wesselborg and Stork, 2015; Nishida et al., 2015.) Themembrane becomes enclosed around its cargo to create a double-membraned autophagosome (Nishida et al., 2015; Nemchenko et al.,2011; Eskelinen et al., 2005). Material degraded throughmacroautophagy includes defective mitochondria, ribosomes,peroxisomes, endoplasmic reticulum and additional membranestructures, glycogen, lipid granules and other cytosolic aggregates(Kovsan et al., 2010; Kundu et al., 2008; Kuma and Mizushima,2010; Schneider and Cuervo, 2014; Shires and Gustafsson, 2015;Nishida et al., 2015). The targeted material might be identified bycargo-recognizing proteins, which bind to the cargo and amembranecomponent of the developing autophagosome such as LC3(Schneider and Cuervo, 2014; Wild et al., 2014). For example,PTEN-induced kinase 1 (PINK1), Parkin and p62 (also known asSQSTM1) are involved in identifying mitochondria for degradation,whereas p62 is additionally involved in recognizing lipid droplets(Jin and Youle, 2012; Schneider and Cuervo, 2014). Mutations inPINK1 and Parkin are associated with Parkinson’s disease, in whichcells exhibit defective mitochondrial turnover (Jin and Youle, 2012;Schneider and Cuervo, 2014). LC3, a molecule frequently used as amarker of autophagy (and the mammalian homolog of Atg8), hasbeen thought to be required for expansion of the isolation membraneand autophagosome formation, although exceptions have beenfound (Kishi-Itakura et al., 2014; Nishida et al., 2009). Duringautophagy, LC3 is typically converted from the soluble form LC3-Ito the lipidated form LC3-II (Klionsky et al., 2007). However,certain Atg-knockout cells (ATG5 and ATG3 knockouts) canaccumulate expanded isolation membranes without convertingLC3-I to LC3-II (Kishi-Itakura et al., 2014), and it has also beenfound that in some alternative macroautophagy processes the LC3-Ito LC3-II conversion does not necessarily occur (Nishida et al.,2009). Additionally, findings arising from measurement of theconversion of LC3-I to LC3-II to determine autophagy activity andrates have been controversial (Klionsky et al., 2007), altogethermaking LC3 conversion a questionable marker of autophagy.Following this, the outer membrane of an autophagosome fuses

with a lysosomemembrane in a LAMP-2-dependentmanner to forman autolysosome (Endo et al., 2015); therein, the cargo of the innermembrane is degraded by the digestive enzymes of the lysosome,generating amino acids and lipids that are transported to the cytosolto be reused (Nishida et al., 2015). Alternatively, an autophagosomemight fuse with an endosome (a vesicle formed from the plasmamembrane of the cell) in a LAMP-2-dependent manner to form anamphisome, which then fuses with a lysosome. Because the LAMP-2B isoform is thought to be involved in macroautophagy, theselysosome fusion processes – which ensure proper autophagosomefunction and maturation – are likely to be perturbed upon loss offunction of LAMP-2B. It is worth noting, however, that the presenceof excessive autophagic vacuoles in individuals with Danon diseasedoes not necessarily indicate that LAMP-2B deficiency is truly

causal. Although LAMP-2 is primarily localized to the lysosomemembrane, it is additionally found in the membranes of earlyendosomes, late endosomes and amphisomes, as well as in plasmamembranes (Endo et al., 2015). The presence of LAMP-2 on theinner surface of lysosomemembranesmight also help tomaintain anacidic environment in the lysosome and prevent lysosomal digestiveenzymes from auto-digesting cellular cytoplasmic components(Endo et al., 2015; Saftig et al., 2001). Interestingly, through itscytoplasmic tail, LAMP-2B (but not LAMP-2A) has been found tointeract with the transporter associated with antigen processing like(TAPL; also known as ABCB9) protein, which transports peptidesin the cytosol to the lysosomal lumen (Demirel et al., 2012).Although this interaction does not appear to affect the subcellularlocalization or transportation of the peptides, LAMP-2B mightinstead act to increase the half-life of TAPL (Demirel et al., 2012).Other major players in the fusion of autophagosomes to lysosomesare the soluble NSF attachment protein receptor (SNARE) proteins;these are not known to interact with LAMP-2. In particular, theautophagosomal SNARE protein syntaxin 17 (STX17) has a keyrole in this fusion process and localizes to the outer membrane ofautophagosomes (but is not found in isolation membranes);for details on these and other fusion factors whose discussion isbeyond the scope of this Commentary, see Nishida et al. (2015),Itakura et al. (2012), Schneider and Cuervo (2014), Shen andMizushima (2014). It is possible that the ubiquitin–proteasomesystem and microautophagy are upregulated to compensate formacroautophagy and/or CMA blockage, which could be a potentialtherapeutic option to explore for Danon disease individuals (Masseyet al., 2008).

Like macroautophagy, CMA can be activated by cellular stress,such as oxidative stress, starvation and UV exposure, but CMA ismuch more selective than macroautophagy because it only targetsspecific proteins (Massey et al., 2008; Mrschtik and Ryan, 2015;Schneider and Cuervo, 2014). CMA uses the cytosolic chaperoneheat shock cognate protein 70 (Hsc70; also known as HSPA8),which recognizes proteins containing a specific amino acidtargeting motif (KFERQ-like). Hsc70 binds to these proteins, andthe chaperone–substrate complex is transported to the lysosome,where it interacts with LAMP-2A on the lysosomal membrane totransport the target protein across the membrane and into thelysosomal lumen for degradation. For more details on CMA that arebeyond the scope of this Commentary, see Kaushik and Cuervo(2012), Schneider and Cuervo (2014).

Mouse models of Danon disease – similarities anddifferencesA LAMP2 knockout mouse model of Danon disease has been usedin multiple studies with the aim to better characterize the disease;however, although these mice display many features similar to thoseseen in humans with Danon disease, clear differences have alsobeen reported. Based on amino acid sequence homology, the shortcytoplasmic tail, which might be involved in receptor-mediatedfunctions (i.e. CMA or macroautophagy), is highly conservedbetween humans and mice for the LAMP-2A and LAMP-2Bisoforms [with 82% (9 out of 11) residues being identical for theLAMP-2A isoforms, and 91% (10 out of 11) residues beingidentical for the LAMP-2B isoforms] (Fig. 2B). However, thelumenal portion of LAMP-2 (exons 1–8) is less conserved betweenspecies [with 61% (226 out of 368) identical and 75% (276 out of368) similar or identical residues, and 2% (10 out of 368) gaps](Fig. 2A). Like humans, mice also have a LAMP-2C isoform(Eskelinen et al., 2005).

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Phenotypically, both humans with Danon disease and Lamp2-knockout mice have increased mortality rates, with 50% of micedying between the age of 20 and 40 days (Saftig et al., 2001; Tanakaet al., 2000); all mice show an accumulation of autophagic vacuolesin cardiac and skeletal muscle cells (Tanaka et al., 2000; Stypmannet al., 2006; Saftig et al., 2001; Yang and Vatta, 2007; Eskelinen,2006). Also, similar to the human pathology, Lamp2-knockout micedevelop severely reduced cardiac contractility, with a reducedejection fraction and cardiac output, as well as increased ratios ofheart weight to total body weight, important features forrecapitulating the hypertrophic cardiac phenotype characteristic ofDanon disease (Tanaka et al., 2000; Stypmann et al., 2006;Eskelinen, 2006). However, the cardiac phenotype in humans seemsmore severe overall; the vacuolation in human cardiomyocytesappears to be more pronounced than it is in Lamp2-knockout mice.This is relevant because cardiomyopathy is usually the majordefining characteristic of the human disease (Saftig et al., 2001). Bycontrast, skeletal muscle physiologically appears to be affected tosimilar extents in the mouse model and human disease; myofibers inboth systems are degenerative, with proximal muscles being moreseverely impacted than distal ones (Saftig et al., 2001). Similar toindividuals with Danon disease who develop vision problems andmild cognitive defects, some neuropathological studies of Lamp2-knockout mice have also suggested that the central nervous systemis impacted, specifically manifesting as impaired learning (likely tobe caused by hippocampal dysfunction due to disrupted lysosomalactivity) and motor deficits (Rothaug et al., 2015). However, earliermouse studies have not reported abnormalities of the centralnervous system (Saftig et al., 2001; Endo et al., 2015).Beyond the cardiac and skeletal muscle phenotypes, there are

potential differences in other organs affected. Lamp2-knockoutmice have been reported to have excessive accumulations ofautophagic vacuoles in organs that are not typically affected inindividuals with Danon disease, specifically the liver, pancreas,spleen, thymus and kidneys (Saftig et al., 2001; Tanaka et al., 2000;Yang and Vatta, 2007). Lamp2-knockout mice also displayperiodontitis due to impaired neutrophils being unable toeffectively eliminate bacterial pathogens, which is likely to becaused by reduced fusion of late endosomes and/or lysosomes withphagosomes (Beersten et al., 2007). Although in some Danondisease individuals, increases in the activity of liver enzymes andautophagosome accumulations in hepatocytes have been reported,the autophagic function of hepatocytes is much more severelyimpacted in mice (Saftig et al., 2001; Yang and Vatta, 2007; Tanakaet al., 2000). Overall, the mouse model might represent a moresevere pathology than that seen in individuals with Danon disease interms of the number of different organs affected, although thecardiac phenotype in humans appears to be more severe, which isnoteworthy because this is the primary cause of mortality in people(Saftig et al., 2001; Endo et al., 2015). However, it has beenproposed that defects in other tissues of individuals with Danondisease might have been overlooked as the focus was on examiningthe severe cardiac dysfunction (Endo et al., 2015).In terms of molecular and cellular characterization, affected

tissues of the Lamp2-knockout mice share many similarities withthose of individuals with Danon disease, and examinations of celllines derived from mice have afforded insights into the molecularpathogenic mechanisms of Danon disease. As seen in humans,glycogen accumulates within the affected mouse model cells, bothinside and outside of autophagic vacuoles (Tanaka et al., 2000).Affected mouse cardiac cells have been reported to frequentlycontain a single mitochondrion in their autophagic vacuoles

(Eskelinen, 2006), which reflect the dysfunctional mitochondrialbehavior observed in human iPSC-CMs derived from individualswith Danon disease (Hashem et al., 2015). In the accumulatedautophagosomes in pathogenic mouse hepatocytes, fusion betweenthe autophagosomes and lysosomes appears to be impaired, whereasfusion between endosomes and autophagosomes does not appear tobe altered (Eskelinen, 2005; Tanaka et al., 2000). Of note, Lamp2-deficient mice accumulate cholesterol in their liver (Schneede et al.,2011) and brain (Rothaug et al., 2015). This cholesterol defect hasbeen further characterized in Lamp2-deficient mouse embryonicfibroblasts (MEFs), which accumulate unesterified cholesterol inlate endosomes and/or lysosomes (Eskelinen et al., 2004; Eskelinen,2006). This accumulation could be rescued by the lumenal domainand membrane-proximal part of LAMP-2 (in a mechanismindependent of CMA), suggesting that these regions of LAMP-2might have a crucial role in the transport of unesterified cholesterol.Specifically, unesterified cholesterol is generated by endogenoussynthesis in the cytosol or through low-density lipoprotein (LDL)uptake, and becomes esterified in the endoplasmic reticulum.LAMP-2 might be important for cholesterol transportation from thecytosol to the endoplasmic reticulum (Schneede et al., 2011).

Concluding remarksAlthough the progression of Danon disease in individuals hasbecome better characterized clinically, our understanding of theunderlying pathological molecular mechanisms remains limited. Inparticular, improving our knowledge of the role of LAMP-2 inmacroautophagy, and potentially in other intracellular vesicletransport mechanisms that the LAMP-2B isoform might beinvolved in, might help us to better understand the disease and,consequently, to developmore targeted and effective treatment plans.Lamp2-deficient mice have been valuable tools in investigating thisdisease, although as discussed here, caution should be taken whendrawing parallels between these models and the human pathologybecause there are clear differences, such as a potentially more severecardiac phenotype in humans. The recent development of human-derived iPSC-CMs offers a promising way to investigate themolecular mechanisms under controlled conditions, although thiscell-based system is clearly limited in recapitulating whole-animalphysiological conditions. Moving forward, researchers are likely toneed to utilize both animal and cellular models in order to bestunderstand the molecular aspects of Danon disease and rationallydevelop effective treatments based on the knowledge gained.

AcknowledgementsThe authors would like to thank Andrew J. Bonham (Metropolitan State University ofDenver, Denver, CO), for critical reading of the manuscript. The authors would like toalso thank Carmen C. Sucharov (University of Colorado Anschutz Medical Campus,Aurora, CO) and Eric D. Adler and Sherin I. Hashem (University of California SanDiego, San Diego, CA) for their unpublished data contributions.

Competing interestsThe authors declare no competing or financial interests.

FundingSupportedbyNational InstitutesofHealth [grant number1R01HL109209-01A1]; andaTrans-Atlantic Network of Excellence grant from Fondation Leducq (LeducqFoundation) [grant number14-CVD03].Deposited inPMC for releaseafter 12months.

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