Lysosomal Storage Diseases-Regulating Neurodegeneration.

Autophagy is a complex pathway regulated by numerous signaling events that recycles macromolecules and can be perturbed in lysosomal storage diseases (LSDs). The concept of LSDs, which are characterized by aberrant, excessive storage of cellular material in lysosomes, developed following the discovery of an enzyme deficiency as the cause of Pompe disease in 1963. Great strides have since been made in better understanding the biology of LSDs. Defective lysosomal storage typically occurs in many cell types, but the nervous system, including the central nervous system and peripheral nervous system, is particularly vulnerable to LSDs, being affected in two-thirds of LSDs. This review provides a summary of some of the better characterized LSDs and the pathways affected in these disorders.

Introduction

Cellular homeostasis is essentially a balancing act between anabolic and catabolic processes. Eukaryotic cells primarily use two distinct mechanisms for large-scale degradation of macromolecules and intracellular organelles: proteasomal degradation and autophagy. However, only autophagy, which can be further subdivided into macroautophagy, microautophagy, and chaperone-mediated autophagy, has the capacity to degrade entire organelles.1 Here, we focus on macroautophagy, hereafter termed simply as autophagy, and its important physiological role in human health and in neurodegeneration, including lysosomal storage diseases (LSDs). We also discuss the possibility of autophagic regulation by various signaling pathways (eg, extracellular signal-regulated kinase [ERK], microtubule-associated protein kinase [MAPK], Akt, target of rapamycin [TOR], and AMP-activated protein kinase [AMPK]) and other mechanisms (eg, Ca2+ levels). We begin by outlining the complex steps required to complete autophagy, then address the neurodegeneration that has been described in multiple LSDs, and finally examine several LSDs individually and in more detail.

Autophagy is a pathway required for the degradation of cellular macromolecules.2,3 During autophagy, double membrane-bound vesicles (autophagosomes) isolate cytosolic material destined for degradation. Subsequently, autophagosomes fuse with late endosomes to form amphisomes.4,5 Amphisomes then coalesce with lysosomes, leading to the formation of autolysosomes (Fig. 1). Because lysosomes contain degradatory enzymes, the contents of amphisomes are broken down following autolysosome formation, with the produced metabolites partly feeding into pathways to satisfy the cell’s energy demands.4,5 Downregulation of autophagy leads to accumulation of misfolded proteins and is speculated to be involved in chronic or late-onset diseases, such as neurodegenerative diseases, including Alzheimer’s disease (AD; characterized by two abnormal structures: amyloid plaques consisting of largely insoluble toxic β-amyloid peptides and intraneuronal fibrillary tangles/aggregates composed of highly phosphorylated forms of the microtubule-associated protein tau), Parkinson’s disease (PD; well characterized by the accumulation of α-synuclein and ubiquitin into intracytoplasmic inclusions known as Lewy bodies), and Huntington’s disease (HD; toxic oligomers and aggregates of mutant huntingtin protein that are not properly cleared accumulate).6–8 These aforementioned diseases have been extensively covered in the literature, and several excellent reviews focusing on them as neurodegenerative diseases caused by aberrant autophagy exist.8,9 Therefore, we focus on rarer diseases involving aberrant autophagy, the LSDs.

Figure 1

General organization and function of some integral membrane and soluble proteins important for lysosomal function, vesicle fusion, pH regulation, and calcium homeostasis (eg, MCOLN1); cholesterol homeostasis (eg, NPC1 and NPC2); and lysosomal function, vesicle fusion, and cholesterol homeostasis (eg, LAMP2). For MCOLN1, the model also illustrates how mTOR, AMPK, and MCOLN1 possibly interact. When active, AMPK inhibits mTOR (or its downstream effector molecule S6 kinase 1) activity, which in turn modulates MCOLN1 activity in a feedback loop (ie, inactive/less active mTOR leads to increased MCOLN1 activity; however, activating MCOLN1 increases autophagy, leading to increased amino acid production and activating mTOR—a feedback loop that regulates/modulates mTOR, AMPK, and MCOLN1 activities).14,173

Since the discovery of the lysosome by De Duve,10,11 more than 60 distinct LSDs have been described, with the collective incidence of their occurrence estimated to be ~1:5000 worldwide.12,13 In general, LSDs can be described as a subgroup of inborn errors of metabolism and primarily result from a deficiency/defect of one or more lysosomal enzymes involved in macromolecule degradation (several excellent reviews, which will be cited herein, exist14–18). However, in some LSDs, the exact function of the mutated protein(s) has yet to be determined.18,19 Roughly two-thirds to three-quarters of LSDs have some neurological component, affecting multiple brain regions but dependent on the specific disease type. A few examples of LSDs that are associated with central nervous system (CNS) and peripheral nervous system (PNS) pathology include Gaucher’s disease, Krabbe disease, Sandhoff disease (SD), Niemann–Pick type C (NPC), mucolipidoses, and the group of neuronal ceroid lipofuscinoses (NCLs; commonly referred to as Batten disease).20 This review highlights select LSDs that affect the CNS and PNS, briefly addresses the neuropathology associated with these disorders, and provides some mechanistic detail on the presumptive causes leading to the disorders, focusing on therapeutic strategies and/or targets. The various enzymes/proteins that are mutated in the LSDs discussed in this review will be dissected since they play a critical role in lysosomal homeostasis/function. However, an intriguing finding is that not all LSDs have a dramatic CNS pathology, which brings into question the functional importance of mutated genes in the brain (and in neurons in general) compared to other organs.18

Neurodegeneration in LSDs

While the mechanistic details behind the neural degeneration observed in many LSDs are not completely understood, the abnormal accumulation of lysosomal storage material due to defective degradation processes was originally thought to contribute to neuronal loss in LSDs21–23 and other neurodegenerative disorders typified by protein aggregation, such as AD and PD.24,25 However, this reasoning has more recently been called into question based on the finding that lysosomal storage material accumulation is typically widespread in neurons throughout the brain, even though only select neuronal populations are affected.18 Nevertheless, most neurons are postmitotic and unable to eliminate unwanted/damaged organelles and macromolecules by dividing. Therefore, neurons must heavily rely upon functional lysosomes/autophagy to efficiently clear these molecules. Autophagic defects have been reported in several LSDs, including Pompe disease, multiple sulfatase deficiency (MSD), NPC, Gaucher’s disease, and NCLs,23 and are suggested to contribute to neurodegeneration.18

Data from electron microscopy (and other imaging) studies and biochemical analyses of cell lines and tissues from LSD mouse models also support the idea that mitochondrial dysfunction in neurons is responsible for various LSDs, including Gaucher’s disease, MSD, NPC, and mucopolysaccharidoses.26–28 In addition, perturbed mitochondrial Ca2+ homeostasis and/or release has also been observed in the aforementioned LSDs, including decreased Ca2+ buffering capacity, reduced ATP production, and mitochondrial fragmentation.21,29–31 In fact, a reduction in mitochondrial membrane potential and a concomitant decrease in ATP yield have already been shown in a NPC1 mouse model.26 Decreased oxygen consumption and mitochondrial electron transport chain enzymes have also been reported in neurons from a mouse model of juvenile NCL (JNCL).32 Similarly, enlarged mitochondria have been observed in a neuronal cell line derived from JNCL mice;33 however, it should be noted that this particular neuronal cell type is not lost during the progression of the disease. Therefore, mitochondrial abnormalities such as these may represent a common feature of LSDs, indicating that an energy deficit could be one of the contributing mechanisms responsible for neurodegeneration.18

In terms of the potential molecular mechanisms whereby LSDs alter and affect the function and survival of neurons, other neurodegenerative diseases can serve as examples. For example, various signaling pathways are known to contribute to reactive astrocytosis (astrocyte activation) in acute and chronic neurological conditions.34,35 These include Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling and ERK1/2 phosphorylation.36 Notably, JAK/STAT3 activation has been observed in the mouse model of SD, which is mediated by tumor necrosis factor (TNF) α production. Inhibition of TNFα in this double knockout mouse significantly inhibits astrocyte activation and reduces neuronal death. In these mouse models, such changes coincide with a significantly increased lifespan, enhanced coordination, and improved neurological function. Interestingly, these improvements in the mouse model of SD are not accompanied by alterations in ganglioside accumulation in neurons.37 Similarly, increased ERK phosphorylation has also been shown in a model of infantile NCL (INCL) where reactive astrocytes are a prominent feature and associated with aggressive neurodegeneration.18,38

Demyelination (either in the CNS or in the PNS), which also ultimately impacts neuronal survival and function, is another hallmark of LSDs.39 Specialized neuroglia, nonneuronal cells (oligodendrocytes and Schwann cells) coat axons in the CNS and PNS, respectively, with their cell membrane, forming a membrane known as myelin, producing the myelin sheath.39,40 This sheath then provides insulation to the axon so that electrical signals can propagate more efficiently.39,40 In a number of LSDs, eg, Krabbe disease, MSD, and NPC, myelination is aberrant (either delayed or abnormal), resulting in demyelination and subsequently severe neurological impairments (as will be further discussed later).18,40

LSDs Associated with Nonmembrane-bound Lysosomal Hydrolases

Gaucher’s disease (a sphingolipidosis)

Gaucher’s disease is a prototypical LSD (prevalence of ~1:50,000 in the general population) caused by mutations in the glucocerebrosidase (GBA1) gene, a lysosomal enzyme responsible for the degradation of glucocerebroside, which is an intermediate in glycolipid metabolism.41–43 Hundreds (nearly 300) of GBA mutations have been identified and include missense, nonsense, and frameshift mutations. Collectively, these mutations have been linked to three forms of Gaucher’s disease, types 1–3.44,45

Type 1, typically referred to as adult or visceral Gaucher’s disease, is generally late onset and represents the most common form, with an increased ethnic incidence among Ashkenazi Jews, a prevalence as high as 1:850 has been previously reported.46 Type 2 has the earliest onset (approximately three to six months of age), with death usually occurring by two years. Type 3 is a juvenile disease with an onset in early childhood. As a result of GBA deficiency, lysosomes accumulate several glycolipids, including glucocerebroside and glucosylsphingosine.47,48 The major cell type affected in Gaucher’s disease is the macrophage, and resident macrophage populations within the spleen and liver have perturbed homeostatic functions.47 As a result, there is marked spleen enlargement (splenomegaly), which destroys hematopoietic cells leading to anemia.49 The neuronopathic forms of Gaucher’s disease (types 2 and 3, which are acute and chronic, respectively) are also characterized by microglial proliferation, astrocytosis, and a robust neuroinflammatory response and have no available treatment.50,51 Currently, it is not well understood why only particular brain regions are selectively targeted given the ubiquitous expression of GBA; however, it is clear that storage material accumulation is not the primary deciding factor, ie, a series of secondary events, including neuroinflammation and neurodegeneration, are apparently triggered by a certain threshold of accumulation, resulting in neuronal death but only in specific brain areas where the neurons are intrinsically more sensitive to the inflammatory response.48,50,52

A mouse model of Gaucher’s disease, where GBA is selectively deleted in neurons and glia, results in increased expression of the lysosomal enzyme cathepsin D;53 this may represent a compensatory mechanism to offset GBA deficiency. Compared to wild-type mice, the expression of brain-derived neurotrophic factor and nerve growth factor is reduced in the cerebral cortex, brainstem, and cerebellum of Gaucher mice, and ERK1/2 expression is downregulated in neurons from Gaucher mice, which correlates with a decreased number of neurons.54 Because brain-derived neurotrophic factor and nerve growth factor protect neurons and activate the MAPK pathway,55–57 these results suggest that a reduction in neurotrophic factors could be involved in neuronal loss in Gaucher’s disease.18,54

Fabry’s disease and GM1 gangliosidosis (sphingo-lipidoses)

GM1 gangliosidosis is an autosomal recessive lysosomal lipid storage disorder caused by mutations of the lysosomal β-galactosidase and results in the accumulation of GM1 ganglioside. The disease phenotype is characterized by severe CNS (primarily neurons but astrocytes may also be vacuolated) dysfunction and skeletal dysplasia.58 Increased basal expression of the autophagosome marker microtubule-associated protein light chain 3 (LC3-II) is observed in several sphingolipidosis models, including GM1 gangliosidosis58 and Fabry’s disease,59 while an increased number of autophagosomes (detected by the LC3 marker), elevated Beclin 1 levels, and dysfunctional (both morphologically abnormal and with a decreased membrane potential) mitochondria are specifically observed in brains from GM1 gangliosidosis mice.58 The Akt–mTOR and Erk signaling pathways are also activated in the GM1 mouse model,58 thereby inducing autophagy; however, detailed mechanistic information is still unavailable.60

In Fabry’s disease, deficiency of the lysosomal enzyme α-galactosidase A results in an accumulation of its substrate, globotriaosylceramide (Gb3), throughout the body, leading to neurological manifestations of disease in both the PNS and CNS, including Schwann cells and dorsal root ganglia together with deposits in CNS neurons.61 Measurement of LC3-II in cultured cells from patients with Fabry’s disease reveals increased basal levels when compared with wild-type cells and, as might be expected, a larger increase in response to starvation. Treatment of starved Fabry’s disease cells with lysosomal protease inhibitors reveals a block/impairment in autophagic flux, demonstrating a more severe disruption of degradation through macroautophagy than that observed in other sphingolipidoses. In addition, increased p62/SQSTM1 and ubiquitin staining in renal tissues and in cultured fibroblasts from patients with Fabry’s disease further supports impaired autophagic flux.59 For Fabry’s disease and other sphingolipid storage diseases, defining where and how this impairment in autophagic flux occurs and establishing the extent to which alterations in macroautophagy contribute to the disease phenotype remain important research goals.18

SD, a GM2 gangliosidosis (a sphingolipidosis)

SD is a rare autosomal LSD caused by a deficiency in β-hexosaminidases A and B and results in the excessive lysosomal accumulation of GM2 gangliosides and oligosaccharides.62 There are three clinical subtypes of SD, namely infantile, juvenile, and adult onset. The infantile form is the most aggressive—typically presenting between two and nine months of age—with death occurring before three years. The juvenile form of SD is less common than the infantile variant, with clinical symptoms evident between the ages of 3 and 10 years, which include organomegaly, bone deformations, and CNS (ballooned neuronal cells, astrocytes, and histiocytes) manifestations, such as speech disabilities, cerebral ataxia, and severe psychomotor disturbances.63 Neuropathological abnormalities associated with SD include prominent brain atrophy and dilatation. Histologically, neurons harbor membranous cytoplasmic bodies formed by the accumulation of GM gangliosides and other lipopigments in the lysosome.62 An earlier report examining primary astrocytes isolated from a mouse model of SD demonstrated an increased proliferation that was associated with elevated ERK phosphorylation and sphingosine-1-phosphate (S1P) synthesis.64 These changes were dependent on GM2 ganglioside accumulation within the lysosome. In addition, a direct relationship between S1P metabolism and reactive astrocytosis is indicated by the mouse model of SD, where the deletion of sphingosine kinase (which synthesizes S1P) or S1P receptor reduces astrocyte proliferation and, therefore, reactive astrocytosis.65 Interestingly, S1P has recently emerged as a key neuroinflammatory mediator in multiple sclerosis and is being explored as a potential therapeutic target to attenuate disease severity.18,66–68

SD shares many features with other neurodegenerative disorders, such as increased reactive astrocyte pathology,69 and activating the JAK2/STAT3 pathway using the inflammatory factor TNFα may be a mechanism for astrocyte activation in the disease.37 Bone marrow transplantation experiments have revealed that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. Therefore, TNFα can presumably function as a neurodegenerative cytokine, mediating astrocytic pathology and neuronal cell death in SD, and as a potential therapeutic target to attenuate the observed neuropathology.37

Krabbe disease (a sphingolipidosis)

Krabbe disease, also known as globoid cell leukodystrophy, results from β-galactocerebrosidase deficiency, the enzyme catalyzing the hydrolysis of galactose from several sphingolipids to generate ceramide and sphingosine.70,71 β-Galactocerebrosidase loss leads to the accumulation of the glycosphingolipid psychosine, which is toxic—in particular to oligodendrocytes.72 Krabbe disease is an early onset LSD—symptoms typically present at approximately six months of age—and mortality occurs by two years.73 Krabbe disease primarily affects the CNS, resulting in extensive demyelination of the myelin sheath, leading to ataxia, blindness, seizures, and severe dementia.74,75 The neuropathology associated with Krabbe disease has been attributed, in large part, to the abnormal accumulation of psychosine in the brain, which will be discussed further below.76–78 Metabolic alterations in astrocytes have been reported in the mouse model of Krabbe disease, the twitcher mouse, and include increased glutamine levels and upregulation of lactate-specific transporters.79 Microglial activation has also been reported in patients with Krabbe disease, which is consistent with a prominent neuroinflammatory response.80 This inflammatory response likely results from cell loss and the release of danger-associated molecular patterns from damaged/dying neurons, which can trigger inflammatory pathways and further exacerbate neuronal damage. Indeed, psychosine has also been reported to exert inflammatory and apoptotic effects in glia,81 which correlates well with the increased concentration of psychosine in the brains of patients with Krabbe disease and in the respective animal model, the twitcher mouse.18,82–84 Several mechanisms of action have been proposed for psychosine in Krabbe disease:

Lysosphingolipids, such as psychosine, are potent reversible inhibitors of protein kinase C (PKC).85 It is well-known that PKC is activated by the lipid diacylglycerol, which is generated from phosphatidylinositol bisphosphate in signal transduction pathways mediated by phospholipase C. As mentioned earlier, psychosine accumulates in Krabbe disease, leading to the apoptosis of neurons and astrocytes.86–88 It is, therefore, of interest that Schwann cells from twitcher mice are 10-fold more sensitive to staurosporine—a PKC inhibitor—than normal cells, indicating a preexisting inhibition of PKC— possibly by psychosine. Interference with PKC-mediated growth factor signaling could therefore partially account for the loss of myelin-producing cells in Krabbe disease.

In oligodendrocytes, insulin-like growth factor 1 (IGF-1) acts through the activation of the antiapoptotic PI3K-Akt/Protein kinase B (PKB) or the MAPK/Erk1/2 signal transduction pathways, and in murine oligodendrocyte precursor cells, psychosine leads to a dose-dependent decrease in both Akt and ERK1/2 phosphorylation accompanied by an activation of caspase-3, resulting in apoptosis. When psychosine-treated cells are exposed to high doses of IGF-1, Akt phosphorylation, and to a lesser extent Erk1/2 phosphorylation, is restored. This leads to a reduced cleavage of caspase-3, resulting in a reduced apoptotic rate in oligodendrocyte precursor cells.89 Thus, the inhibition of IGF-1 mediated antiapoptotic signaling pathways by psychosine may be one reason for the death of oligodendrocytes in Krabbe disease.

Another major target of psychosine is phospholipase A2, which cleaves the membrane lipid phosphatidylcholine into lysophosphatidylcholine and arachidonic acid. Both products are biologically highly active lipids involved in numerous physiological and pathophysiological reactions, with the injection of lysophosphatidylcholine into the brain inducing demyelination in vivo.90

Psychosine also reduces AMPK activity. AMPK, which is considered as an important enzyme in the regulation of glucose and lipid metabolism, senses cellular energy levels and maintains the balance between ATP production and consumption.91,92 In a status of low energy, it is activated, switching off anabolic pathways and activating catabolic pathways and vice versa.93,94 Exposing cells to psychosine downregulates AMPK activity, leading to a preponderance of biosynthetic pathways in treated cells, eg, oligodendrocytes treated with psychosine display an enhanced synthesis of fatty acids and cholesterol, while β-oxidation as a catabolic pathway is inhibited. Thus, psychosine may also influence the energetic status of a cell by modulating the master switch AMPK, affecting the energy balance.95 The inhibition of this kinase by psychosine favors energy-consuming pathways over energy-generating pathways, and the resulting lower energy load could also contribute to oligodendrocyte loss.

Glycogen storage disease type II (also known as Pompe disease) (a glycogenosis)

Though first discovered more than 80 years ago,96 Pompe disease would only later (~30 years later) be the first recognized LSD.97 The disease is caused by a deficiency in acid maltase, also known as acid α-glucosidase, leading to the accumulation of glycogen in the lysosome, lysosomal enlargement, a dramatic expansion of all vesicles of the endocytic/autophagic pathways, and a slowdown in the vesicular trafficking in the overcrowded cells, ultimately leading to profound muscle and nerve cell damage.98–100 Clinical heterogeneity of the disease is a well-established phenomenon.101,102 In the most serious infantile form, the disease leads to profound weakness and heart failure and, if left untreated, causes death within one year.23,103–105 However, even in the milder late onset form, the illness is extremely debilitating, with patients eventually becoming confined to a wheelchair or bedridden, and many die prematurely from respiratory failure.23,103–105

Only recently has enzyme replacement therapy using recombinant human α-glucosidase designed to supplement the defective enzyme been approved for all forms of the disease. This therapy stemmed from a straightforward approach to explain the pathogenesis of the disease that the progressive enlargement of glycogen-filled lysosomes would lead to lysosomal rupture and to release of glycogen and other toxic substances into the cytosol.23 The assumption was that early treatment, initiated before lysosomal integrity was compromised, would reverse this pathogenic cascade. However, this assumption is apparently only partially correct—cardiac muscle responds very well to therapy, but skeletal muscle does not. In particular, this poor muscle response to the therapy has led to a revisiting of the pathogenesis of the disease, and more recently modulating transcription factor EB has been proposed as a new approach to circumvent the problem of inefficient enzyme delivery by exploiting the ability of lysosomes to expel their contents into the extracellular space, providing clearance of the stored material.106,107 Indeed, transcription factor EB overexpression in Pompe disease muscle has been demonstrated to alleviate autophagic pathology—it promotes the formation and removal of excessive autophagic vacuoles. Thus, a promising new drug target for treating Pompe disease does exist.107

Multiple sulfatase deficiency (a mucopolysacchari-dosis/sulfatidosis)

Mucopolysaccharidoses represent a substantial proportion (~25%) of all LSDs.22 MSD is caused by a mutation in sulfatase-modifying factor 1 (sumf1), resulting in posttranslational defects in lysosomal sulfatases and the pathological accumulation of mucopolysaccharides and sulfatide.108,109 Therefore, MSD can be more accurately defined as both a mucopolysaccharidosis and a sulfatidosis, or a mucosulfatidosis.110,111 There are three types of MSD, neonatal, late infantile, and juvenile.112 The infantile form of MSD is the most aggressive, with symptoms beginning soon after birth. Clinical manifestations include coarsened facial features, deafness, splenomegaly, and hepatomegaly.113–115 Children with MSD develop a specific neurodegenerative pathology (leukodystrophy), leading to movement disorders and developmental delay with occasional seizures.116,117 The late infantile form is the most common type of MSD. These children have normal cognitive development in early childhood but experience a rapid decline in motor and cognitive abilities that are attributed to progressive leukodystrophy.75 Neuroimaging studies have revealed lesions extending into the brain stem.116 MSD is also typified by extensive demyelination, with the accumulation of cholesterol and galactolipid pigments in the CNS.118 A recent study utilizing a mouse model demonstrated that the targeted deletion of sumf 1 in astrocytes results in severe lysosomal storage material deposition and neuronal loss in vivo.119 A defective autophagic flux has also been demonstrated by the accumulation of autophagy substrates, such as polyubiquitinated proteins and dysfunctional mitochondria, both of which are significantly increased in tissues from MSD and mucopolysaccharidosis type IIIA mice.18,22

Both of these mouse models, MSD and mucopolysaccharidosis type IIIA, present an observed accumulation of autophagosomes resulting from defective/impaired autophagosome–lysosome fusion. This impairment of the autophagic pathway is demonstrated by the inefficient degradation of exogenous aggregate-prone proteins (ie, expanded huntingtin and mutated α-synuclein) in cells from these mice; thus, these LSD models can be defined as autophagy disorders resembling more common neurodegenerative diseases, such as AD, PD, and HD. While there are major differences in the initial steps involved in all these diseases (ie, impaired degradation of polyubiquitinated proteins in LSDs versus expression of aggregate-prone proteins in AD, PD, and HD), they may share common mechanisms, eg, blocked autophagy due to defective fusion between autophagosomes and lysosomes, suggesting the possibility of overlapping therapeutic strategies.22,120

Mucolipidosis type II and mucolipidosis type III

Mucolipidosis type II (MLII) and mucolipidosis type III (MLIII) are autosomal recessive diseases caused by deficiency of the enzyme N-acetylglucosamine 1-phosphotransferase.121–123 This enzyme modifies newly synthesized lysosomal hydrolases by attaching a molecule of mannose-6-phosphate, which functions as a tag for delivery to lysosomes.124 Mutations in GlcNAc-phosphotransferase result in the missorting and cellular loss of lysosomal enzymes and the lysosomal accumulation of storage material.125 MLII is characterized by skeletal abnormalities, short stature, cardiomegaly, and developmental delays, while MLIII is a later onset, milder form of MLII.126 Alterations in autophagy have recently been reported in MLII and MLIII fibroblasts, including the accumulation of autophagosomes, p62/SQSTM1, ubiquitin, and fragmented mitochondria. Additionally, the lysosomal pH of MLII cells has been shown to be higher than that of normal cells.127 In contrast, no variations in the levels of Beclin 1 are observed, suggesting that the formation of autophagosomes is not increased in these disorders.128 Accumulation of LC3-positive structures and ubiquitin aggregates has also been reported in neuronal cells from a patient with MLIII.129 Importantly, inhibition of autophagy restores mitochondrial alterations in MLII and MLIII cells, suggesting that increased autophagy might be detrimental for proper mitochondrial function.128

LSDs Associated with Integral Lysosomal Membrane Proteins

Niemann–Pick type C (a sphingolipidosis)

NPC is caused by mutations in one of the two genes, NPC1 (95% of cases) and NPC2 (5% of cases), which manifest as severe abnormalities in lipid trafficking, eg, NPC1-positive vesicles are believed to be transiently targeted to lysosomes and once there to facilitate clearing of unesterified cholesterol from this organelle, with NPC1 loss of function and the subsequent accumulation of unesterified cholesterol commonly viewed as the principal lesion in NPC.130,131 Both NPC1 and NPC2 are predicted to encode for proteins involved in cholesterol homeostasis, which accounts for the cholesterol accumulation within the lysosome.131–138 NPC affects both peripheral organs and the nervous system, and symptoms include neurological abnormalities, ie, psychomotor disturbances, ataxia, and seizures.130,137 Neuroimaging studies have characteristically revealed diffuse cerebral atrophy and changes within the white matter of the CNS.132 In chronic progressive cases, neurofibrillary tangles similar to the aggregates of hyperphosphorylated tau protein found in AD have also been observed.139 Astrocyte activation is also associated with NPC,140–142 with these cells exhibiting mitochondrial dysfunction.143 Increased levels of IL-1β and increased ApoE, a genetic risk factor for AD, have been reported in animal models of NPC.144 Consistent with this study, increased expression of amyloid precursor protein as well as β- and γ-secretases has been found in reactive astrocytes in mice suffering from NPC disease,145 suggesting an association between NPC and AD.18

Danon disease (a glycogenosis)

Danon disease, which is also known as lysosomal glycogen storage disease with normal acid maltase activity or glycogen storage disease due to lysosomal-associated membrane protein 2 (LAMP2) deficiency, is a lysosomal glycogen storage disease due to LAMP2 deficiency.146,147 The disease is inherited as an X-linked trait and is extremely rare. The disease phenotype is characterized by severe cardiomyopathy and variable skeletal muscle weakness and is often associated with mental retardation.23 Interestingly, Danon disease was the first LSD described in 1981 involving a mutation in a lysosomal structural protein rather than an enzymatic protein,146,147 with the accumulation of autophagic vacuoles in several tissues, particularly in muscle.148 In fact, a patient with Danon disease was initially believed to suffer from another rare LSD, Pompe disease, based on a tissue biopsy. However, the tests for Pompe disease were normal for acid maltase activity.146

Mucolipidosis type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive disorder characterized by acute psychomotor delays, achlorydria, and visual abnormalities, including retinal degeneration and corneal clouding.19,149 Lysosomal inclusions are found in most tissues in patients with MLIV, with the composition of the storage material being heterogeneous and including lipids and mucopolysaccharides forming characteristic multiconcentric lamellae, as well as soluble, granulated proteins.150–155 MLIV is thought to be solely due to mutations in MCOLN1 (mucolipin 1; also known as TRPML1, transient receptor potential MCOLN1), an endolysosomal cation channel belonging to the transient receptor potential superfamily of ion channels.154,156–158 Whole cell patch clamping and native endolysosomal recordings have led to the conclusion that MCOLN1 functions as an inwardly (from lumen to cytoplasm) rectifying channel permeable to Ca2+, Na+, K+, and Fe2+/Mn2+, whose activity is potentiated by low pH.159–163 Although the cellular role of MCOLN1 is still under investigation, the current model suggests that this protein mediates Ca2+ efflux from late endosomes and lysosomes.164,165 Localized Ca2+ release from such acidic stores is required for fusion between endocytic vesicles and to maintain organelle homeostasis, thus suggesting that MCOLN1 is a key regulator of membrane trafficking along the endosomal pathway.

In MCOLN1-deficient fibroblasts, both the degradation of the autophagosome content and the fusion of autophagosomes with late endosomes/lysosomes are delayed compared to control cells.166 This leads to a dramatic accumulation of autophagosomes in the cytosol of MLIV cells as demonstrated by indirect immunofluorescence, LC3-II/LC3-I immunoblot, and electron microscopy.166 This impairment of the autophagic pathway has detrimental consequences for the cell leading to inefficient degradation of protein aggregates and damaged organelles. In particular, accumulation of p62/SQSTM1 inclusions and abnormal mitochondria has been described in MLIV fibroblasts and epithelial cells.28,166,167

A mouse model for MLIV supports late endosomal defects as an important site of dysfunction, and autophagy has also been shown to be defective in primary neurons cultured from these mice.168–170 The MCOLN1−/− mice provide an excellent phenotypic model of the human disease, and all of the hallmarks of MLIV are present in the mice with the exception of corneal clouding.168 Analysis of the brain at eight months shows lysosomal inclusions in multiple cell types, including neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells, with larger inclusions present in neurons, and electron microscopy of primary cerebellar neurons from MCOLN1-deficient mouse embryos demonstrates significant membranous intracytoplasmic storage bodies, despite the lack of gross phenotype at birth.170 Evaluation of macroautophagy in neurons by LC3-II/LC3-I immunoblot also shows increased levels of LC3-II, similar to that observed in human MLIV fibroblasts. LC3-II clearance is also defective, as treatment of the MCOLN1−/− neuronal cultures with protease inhibitors to stimulate autophagy does not result in increased LC3-II levels.170 Demonstration of defective autophagy in MCOLN1-deficient neurons suggests a possible mechanism underlying neurodegeneration, whereby increased protein aggregation and organelle damage lead to autophagic stress and eventual neuronal death.166 The MLIV mouse model provides an important tool for evaluating the complicated interplay between chaperone-mediated autophagy and macroautophagy and their role in neurodegenerative disease.

As mentioned earlier, MCOLN1 is an inwardly rectifying channel permeable to Ca2+, Na+, K+, and Fe2+/Mn2+. Ca2+, in particular, is believed to be significant with regard to the physiological function and regulation of MCOLN1, with the channel releasing luminal Ca2+ to facilitate the Ca2+-dependent fusion of amphisomes with lysosomes. The amino acids generated by the degradation of proteins in the autolysosomes promote TORC1 activation. In addition to inhibiting the initiation of autophagy, activated TORC1 (target of rapamycin complex) also diminishes the endocytosis of MCOLN1.171 In the absence of MCOLN1, fusion of amphisomes and lysosomes is impaired. This leads to a decrease in autophagic flux of amino acids, causing a reduction in TORC1 and upregulation of autophagy. Biochemical (mass spectrometry [MS] and in vitro phosphorylation) and Ca imaging data indicate that the MCOLN1 channel may be directly phosphorylated (at Ser572 and Ser576) and negatively regulated by the TOR kinase, but that AMPK could be involved indirectly through activity on the TOR pathway.172,173 This particular finding validates and expands upon previous studies that have strongly suggested links between TOR and the endocytic system, eg, TOR has been localized to endocytic membranes in yeast, fly, and mammalian cell culture.174–176

However, another study suggests that MCOLN1 activity is negatively regulated by protein kinase A phosphorylation at two different sites (Ser557 and Ser559).172

The Neuronal Ceroid Lipofuscinoses

Though not deemed classic LSDs,17 the NCLs are the most common cause of neurodegeneration among children.177 These disorders typically manifest with blindness, seizures, progressive cognitive defects, and motor failure.178 NCL, commonly referred to as Batten disease, encompasses a family of disorders caused by mutations in the ceroid lipofuscinosis (CLN) genes.177,179,180 Currently, mutations in 14 different CLN genes have been identified that are broadly classified into infantile, late infantile, juvenile (the juvenile form is not a typical LSD, ie, not associated with a typical lysosomal enzyme deficiency16,23,181), and adult onset forms.180,182,183 The childhood forms of Batten disease are characterized by the typical symptoms listed earlier and often result in premature death.183,184 A histopathological hallmark of all NCLs is the lysosomal accumulation of autofluorescent lipopigments and proteins; however, the structural appearance of inclusion material varies according to each disease type.185 Biochemical characterization of storage material has also identified lipophilic proteins, including subunit C of mitochondrial ATP synthase (primarily in JNCL) and sphingolipid pigments (in other forms of NCL).33,186,187 The INCL is the most aggressive, with a life expectancy of two to six years.188,189 INCL is due to a mutation in CLN1, which encodes for palmitoyl protein thioesterase, an enzyme responsible for the cleavage of long-chain fatty acids on several proteins containing cysteine residues.190,191 Late INCL is caused by mutations in CLN2, a lysosomal enzyme (tripeptidyl peptidase I), that cleaves tripeptides from the terminal amine groups of partially unfolded proteins.192,193 JNCL results from mutations in the CLN3 gene.183 The precise function of CLN3 remains unknown; however, based on several functional analyses, CLN3 is predicted to regulate lysosomal acidification, endocytic and vesicle trafficking, and proper maintenance of mitochondrial function.194–196 JNCL is similar to INCL and late INCL and also presents with visual impairment, seizures, and progressive cognitive and motor decline, however, with an advanced onset, typically between 5 and 10 years of age.18,183 In addition, the accumulation of dysfunctional mitochondria, increased expression of LC3-II, and the downregulation of the mammalian target of rapamycin (mTOR) pathway, indicating the activation of autophagosome formation, are detected in JNCL due to mutations of the CLN3 gene, with autophagy likely disrupted at the level of autophagic vacuolar maturation.197,198

Conclusion

LSDs are particularly debilitating metabolic disorders; however, the past several decades have witnessed our ever evolving understanding of their complex biology. At the very least, the study of LSDs has helped highlight vital cellular processes, including calcium homeostasis, pH regulation, apoptosis, autophagy, molecular trafficking, endocytosis, and exocytosis, as well as some of the intra- and intercellular signaling events involved in these processes. This deeper understanding of the biology has broadened the range of therapeutic targets for LSDs and other neurodegenerative disorders, as well as for cancer, eg, targeting LAMP2 (a deficiency of which is the underlying cause of Danon disease) may be a viable treatment option for both AD and HD in the future.199,200 Currently, we have no cure for these diseases, but approved therapies for a handful of LSDs, and many ideas for the development of new treatment options, do exist. Genetic screening programs for at-risk populations, screening of newborns for treatable disorders, provisions for genetic counseling, prenatal diagnosis for at-risk pregnancies, and more recently, preimplantation diagnoses remain the best remedies to decrease, or to at least be better prepared for, the complexities that LSDs present to society.17 The current aim for these disorders is still timely diagnoses to enable the early implementation of available and emerging therapies when available.