Acidic Ca²⁺ stores in neurodegeneration

Abstract

Lysosomes have emerged in the last decade as an immensely important intracellular site of Ca²⁺ storage and signalling. More recently there has been an increase in the number of new ion channels found to be functional on lysosomes and the potential roles that these signalling pathways might play in fundamental cellular processes are being uncovered. Defects in lysosomal function have been shown to result in changes in lysosomal Ca²⁺ homeostasis and ultimately can result in cell death. Several neurodegenerative diseases, from rare lysosomal storage diseases through to more common diseases of ageing, have recently been identified as having alterations in lysosomal Ca²⁺ homeostasis that may play an important role in neuronal excitotoxicity and ultimately cell death. This review will critically summarise these recent findings.

Introduction

In recent years, acidic endo-lysosomal Ca²⁺ stores have emerged as a major component of intracellular Ca²⁺ signalling in mammalian cells(Patel & Cai, 2015). Ca²⁺ is one of the most important signalling molecules inside and outside of cells and is an important component of the triggering of fertilization, cell growth and motility and ultimately cell death(Berridge, Lipp et al., 2000). Within cells Ca²⁺ levels are tightly regulated with nM to low μM levels in the cytosol and high μM to low mM levels within some organelles such as the endoplasmic reticulum and acidic stores such as late endosomes and lysosomes(Lloyd-Evans & Platt, 2011). Changes in cytosolic Ca²⁺ levels, via Ca²⁺ release from intracellular organelles or Ca²⁺ entry from the extracellular fluid, triggers cellular changes(Berridge et al., 2000). Ca²⁺ channels present on the plasma membranes or intracellular organelles act in response to extracellular stimuli (primary messengers) or intracellular second messengers such as nicotinic acid adenine dinucleotide phosphate (NAADP) to either trigger Ca²⁺ entry into the cell or Ca²⁺ exit from organelles(Galione & Churchill, 2002). In order to restore cytosolic Ca²⁺ levels back to their normally low levels a variety of pumps and transporters exist that transport Ca²⁺ out of the cell and into intracellular organelles(Brini & Carafoli, 2011, Mekahli, Bultynck et al., 2011). Failure to maintain cytosolic Ca²⁺ levels can lead to continuously elevated Ca²⁺ that can trigger excitotoxicity and cell death(Berridge et al., 2000, Mekahli et al., 2011). One intracellular organelle that appears to regulate global changes in Ca²⁺ signalling and which may act to modulate excitotoxic changes in cytosolic Ca²⁺ is the lysosome(Lloyd-Evans & Platt, 2011, Lloyd-Evans, Waller-Evans et al., 2010). This review aims to highlight the role of the lysosome as a Ca²⁺ store that is involved in the pathogenesis of multiple neurodegenerative diseases.

The need for Ca²⁺ in the endo-lysosomal system

The endo-lysosomal system is an essential series of distinct compartments found in all nucleated cells(Luzio, Pryor et al., 2007). Endocytosis is the process by which material from the extracellular fluid enters the cells, either by binding to receptors or by fluid phase endocytosis, via budding from the plasma membrane. From here, vesicles fuse with the early endosomes and deposited cargo is transported from early endosomes to the late endosome whereas most receptors are recycled back out of the cell via the endocytic recycling compartment. Cargo in the late endosome is usually delivered to lysosomes, the terminal point of the endocytic system, for degradation(Luzio, Parkinson et al., 2009). Lysosomes have an acidic lumen, ranging from pH 4-5 dependent on cell type(Lloyd-Evans, Morgan et al., 2008), and are comprising numerous highly glycosylated transmembrane proteins that form a protective lumenal glycocalyx to prevent self digestion by the lysosomes own high content of acidic hyrolases (Schulze, Kolter et al., 2009). Lysosomes are the main recycling centre of the cell with the products of this degradation of macromolecules being redistributed to endoplasmic reticulum (ER) or Golgi by vesicular transport or pumped into the cytosol via the action of lysosomal membrane transporters(Saftig & Klumperman, 2009). Lysosomes are also essential in the degradation of defective organelles, particularly mitochondria, and mis-folded proteins, all of which enter the lysosomal system through either autophagic vacuole fusion with late endosomes or chaperone mediated autophagy(Platt, Boland et al., 2012).

All of these processes of transport along the endocytic system, membrane recycling and autophagic vacuole fusion are Ca²⁺ dependent processes(Lloyd-Evans et al., 2010, Piper & Luzio, 2004). An early breakthrough in our understanding of how Ca²⁺ regulates endocytic processes was the finding that localised Ca²⁺ release is required for the fusion of lysosomes with late endosomes. Using a fast Ca²⁺chelator, BAPTA, versus the slower acting EGTA, it was found that fusion between purified late endosomes and lysosomes could be inhibited. Furthermore, the amount of Ca²⁺ required to induce fusion between these organelles was in the range of 0.1-10μM. Taken together, this would suggest a rapid localised Ca²⁺ release most likely from a substantial intraorganellar Ca²⁺ store(Pryor, Mullock et al., 2000). Further understanding of the role of Ca²⁺ in regulating endocytosis came from the discovery that the Ca²⁺ dependent phospholipid binding protein Annexin A2, whch mediates cargo delivery between membranes, is required for normal transport of vesicular cargo between early endosomes and late endosomes(Lloyd-Evans et al., 2008, Mayran, Parton et al., 2003). Disruption in endosomal Ca²⁺ handling, the mechanisms of which are discussed in more detail below, can lead to severe disruption in endocytic and cellular function(Lloyd-Evans & Platt, 2011, Lloyd-Evans et al., 2010). Enhanced Ca²⁺ release from lysosomes in response to the second messenger NAADP has been shown to lead to defective endocytosis and recycling of lipids out of late endosomes and lysosomes resulting in their accumulation within enlarged lysosomes(Ruas, Rietdorf et al., 2010). Disruption of Ca²⁺ signalling is also known to lead to defects in autophagic vacuole-lysosome fusion(Gordon, Holen et al., 1993, La Rovere, Roest et al., 2016), another endocytic process which requires localised Ca²⁺ release from lysosomes(Medina, Di Paola et al., 2015), defects in this signalling result in accumulation of autophagosomes and defective mitochondria within cells(Medina & Ballabio, 2015). Furthermore, defects in endo-lysosomal Ca²⁺ release are coupled to changes in pigmentation (Lin-Moshier, Keebler et al., 2014), potentially as a result of altered lysosomal exocytosis . All of these disruptions in the endocytic system are known to occur in neurodegenerative lysosomal storage diseases and more common diseases of ageing(Lloyd-Evans & Haslett, 2016b), and are discussed in more detail below.

Endo-lysosomes are a major intracellular Ca²⁺ store

Although the largest Ca²⁺ store within the cell is the endoplasmic reticulum (ranging from 0.1-1mM (Bygrave & Benedetti, 1996)), it has become apparent in recent years that the endocytic system, especially the so called acidic stores, namely the late endosomes and lysosomes, contain substantial levels of Ca²⁺(Christensen, Myers et al., 2002, Lloyd-Evans et al., 2008). It should however be noted that although the intra-organellar Ca²⁺ content within lysosomes is high (see below), volumetrically lysosomes are predicted as being only 2-3% of the total cellular volume(Thoene, 1992). As such their impact on changing whole cell cytosolic Ca²⁺ levels should be minimal in comparison to ER (>10% of the cellular volume(Voeltz, Rolls et al., 2002)) Ca²⁺ release or extracellular Ca²⁺ influx. However, recent studies have indicated that lysosomal Ca²⁺ release can trigger substantial Ca²⁺ release from the ER which in turn has a global effect on elevating cellular Ca²⁺ levels(Kilpatrick, Eden et al., 2013, Morgan, Davis et al., 2013). The observation of high intra-lumenal lysosomal Ca²⁺ levels explain the origin of the localised elevations in Ca²⁺ that are required for endo-lysosomal fusion and endocytic vesicle transport discussed above(Pryor et al., 2000). Rather than changes in global Ca²⁺ eliciting endocytic transport and recycling, it would appear that this is regulated instead by Ca²⁺ release from endo-lysosomal stores. This was confirmed by studies using dextran conjugated Ca²⁺ chelators endocytosed into lysosomes that induced lysosomal lipid storage and endocytic trafficking abnormalities, indicating that lumenal lysosomal Ca²⁺ is indeed critical for endocytosis(Lloyd-Evans et al., 2008). Whilst extracellular Ca²⁺ levels are high (can range from 1.2-1.8mM in plasma, interstitial fluid and cerebrospinal fluid(Jones & Keep, 1988)), the intra-lumenal Ca²⁺ content of early endosomes has been measured as being in the range of 5-30μM(Gerasimenko, Tepikin et al., 1998). The reason for this discrepancy, which is substantial when one considers the volume of extracellular fluid that continuously enters early endosomes, is the initiation of acidification of the endocytic system at the early endosome level by the action of the H⁺ pumping vacuolar ATPase (v-ATPase)(Gerasimenko et al., 1998, Lelouvier & Puertollano, 2011). This process results in loss of Ca²⁺ from the early endosomal lumen to the cytosol, suggested to be mediated by the transient receptor potential mucolipin (TRPML3) ion channel(Lelouvier & Puertollano, 2011). Furthermore, it has been shown that endocytosis contributes very little to the overall Ca²⁺ content of the acidic stores (Christensen et al., 2002). Using dextran conjugated Ca²⁺ probes we and others have shown that it is possible to specifically load the lysosome with these probes through a pulse/chase experiment. By adjusting the Kd of the Ca²⁺ probe for the pH of the lysosome, and ensuring that a low affinity Ca²⁺ probe is used, it is then possible to measure the intra luminal free Ca²⁺ content of this highly acidic organelle. We have found that although there is some slight differences when comparing cell types, on the whole lysosomal Ca²⁺ appears to be in the range of 500-600μM(Christensen et al., 2002, Lloyd-Evans et al., 2008), indicating that this is the second largest Ca²⁺ store within the cell. How lysosomal Ca²⁺ levels are maintained is discussed in more detail below, and is summarised in Fig. 1, but we are aware that maintenance of the pH gradient is essential as disruption of the v-ATPases leads to expulsion of Ca²⁺ from lysosomes in a similar manner to that which occurs from the ER following inhibition of the ER Ca²⁺ ATPase (SERCA) with thapsigargin(Christensen et al., 2002, Kilpatrick, Magalhaes et al., 2016a).

Figure 1. Maturation of the endocytic system is defined by the indicated changes in pH and Ca²⁺ concentration as well as the expression of ion channels.

Extracellular Ca²⁺ levels is high, yet the process of acidification by the vacuolar ATPase (vATPase) results in Ca²⁺ release out of early endosomes which drives vesicular transport to late endosomes via Ca²⁺ dependent protein machinery such as Annexin A2. Late endosomes and lysosomes have lower pH and higher Ca²⁺ levels which are necessary both for localised events such as vesicular recycling and fusion between late endosomes and lysosomes but also for triggering global changes in Ca²⁺ via triggering of ER Ca²⁺ release. Several new ion channels (indicated and discussed in the text) have been identified on the lysosomal membrane in recent years, highlighting the importance of this Ca²⁺ store to the cell. Green arrows indicate direction of endocytosed cargo (grey circles with black border represent transport vesicles), red arrow indicates fusion between late endosomes and lysosomes, all of these are processes that require local Ca²⁺ release.

Maintenance of endo-lysosomal Ca²⁺ levels; pumps, transporters and channels

Lysosomal Ca²⁺ entry mechanisms

The mechanisms governing Ca²⁺ entry into lysosomes remain largely unknown. Evidence for the presence of an ATPase on lysosomal membranes has emerged from studies into neutrophils and platelets. Platelet dense core granules, organelles very similar to lysosomes, have been shown to contain SERCA3a and it’s inhibition by TBHQ resulted in a reduction in lysosomal Ca²⁺ content(Lopez, Jardin et al., 2008). Furthermore, studies in neutrophils have shown the presence of a high affinity ATP dependent lysosomal Ca²⁺ uptake pump that was depdendent on Mg²⁺(Klemper, 1985). Studies in purified lysosomes however have shown the presence of ATP independent Ca²⁺ entry that can be inhibited by heavy metals including Zn²⁺ and Mg²⁺but not by monovalent ions, is also inhibited by L-cystine and is less efficient when the cytosol is acidified(Lemons & Thoene, 1991). This may provide support for the presence of Ca²⁺/H⁺ exchangers on lysosomal membranes similar to the CAX family which have recently been identified on the lysosomes of non-placental mammals (Melchionda, Pittman et al., 2016) or the solute transporter SLC24A5, a K⁺ dependent Na⁺/Ca²⁺ transporter, has been shown to be important in pigmentation in zebrafish and humans and resides on the surface of melanosomes, a lysosome related organelle(Lamason, Mohideen et al., 2005). Ultimately, the identification of pumps and transporters that could fill the lysosome with Ca²⁺ remains a major fundamental area of research for the lysosomal Ca²⁺ field with the majority of evidence pointing towards some form of transport mechanism reliant on pH(Morgan, Platt et al., 2011). However, a recent study has suggested that Ca²⁺ uptake into lysosomes is not dependent on acidification but instead the ER is utilised, via Ca²⁺ release from InsP3 receptors, to fill the lysosome with Ca²⁺(Garrity, Wang et al., 2016). This study is supported by others indicating that lysosomes selectively sequester Ca²⁺ released from the ER (as opposed to Ca²⁺ entering the cell via store operated pathways) via contact points between these organelles(Lopez Sanjurjo, Tovey et al., 2014, Lopez-Sanjurjo, Tovey et al., 2013). However, these studies also indicate that blocking lysosomal acidification with bafilomycin can perturb the ability of lysosomes to sequester Ca²⁺ released from the ER(Lopez Sanjurjo et al., 2014). All of these studies are in keeping with the findings that alteration in lysosomal pH in familial Alzheimer disease (FAD) cells results in reduced, but not completely diminished, levels of lysosomal Ca²⁺(Coen, Flannagan et al., 2012, Lee, McBrayer et al., 2015). This is described in more detail below but the defect in lysosomal Ca²⁺ in these cells is believed to be activation of TRPML1, a late endosomal/lysosomal ion channel, triggered by abnormal lysoosmal pH incurring a Ca²⁺ leak via TRPML1 out of lysosomes(Lee et al., 2015). This and other studies would therefore suggest that lysosomal pH can maintain endogenous lysosomal Ca²⁺ levels by controlling lysosomal Ca²⁺ uptake from the ER and also triggering lysosomal Ca²⁺ release (Christensen et al., 2002, Lopez Sanjurjo et al., 2014, Morgan & Galione, 2007). It is also evident that Ca²⁺ release from lysosomes can also induce Ca²⁺ release from the ER(Kilpatrick et al., 2013), this is mediated by defined channels described below.

Lysosomal Ca²⁺ release mechanisms

The same is also true for lysosomal Ca²⁺ release, where a plethora of ion channels are now believed to be involved. The two pore channel 2 (TPC2) and the TRPML1 channel are the two best characterised of these ion channels (reviewed in (Patel & Cai, 2015)) but recent evidence has also emerged suggesting that the purinergic ATP activated cation channel P2X4 and the voltage gated Ca²⁺ channel CACNA1a are also present on lysosomes(Cao, Zhong et al., 2015, Huang, Zou et al., 2014, Tian, Gala et al., 2015). For the purpose of this review we will focus on TPC2 and TRPML1 as these two lysosomal ion channels are the ones most associated with neurodegenerative diseases. TPC2 was originally identified as the channel that transports Ca²⁺ out of lysosomes in response to the most potent intracellular Ca²⁺ releasing second messenger NAADP(Brailoiu, Churamani et al., 2009, Calcraft, Ruas et al., 2009), which had previously been shown to target lysosomes(Churchill, Okada et al., 2002). Based on single channel recordings, TPC2 is known to respond reversibly to NAADP under acidic luminal conditions (pH 4.8) and responds to lower concentrations of NAADP when luminal lysosomal Ca²⁺ concentration is higher, suggesting that luminal Ca²⁺ and pH both regulate TPC activity(Pitt, Funnell et al., 2010). The channel has been shown to be capable of transporting Na⁺ (Wang, Zhang et al., 2012) but appears to primarily transport Ca²⁺ (Pitt, Lam et al., 2014) and evidence that TPC2 does not respond to NAADP has been refuted (Ruas, Davis et al., 2015).

In addition to TPC2, the late endosomal/lysosomal system has another well characterised Ca²⁺ permeant channel known as TRPML1. TRPML1 is, at present, the only TRP family member of ion channels exclusively present in the lysosome (TRPM2, a plasma membrane channel, has also been identified on lysosomes(Lange, Yamamoto et al., 2009)) and loss of function is associated with the human lysosomal storage disease mucolipidosis type IV (MLIV) described in detail below. Initially, TRPML1 was believed to be a candidate NAADP responsive channel (Zhang, Jin et al., 2009), but this was shown not to be the case as overexpression of TRPML1 does not enhance NAADP mediated Ca²⁺ release (whereas TPC2 does (Yamaguchi, Jha et al., 2011)). Subsequently, the endogenous ligand for TRPML1 was identified as being phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) (Dong, Shen et al., 2010), which is also capable of modulating TPC2 activity(Jha, Ahuja et al., 2014, Wang et al., 2012). TRPML1 appears to operate in both late endosomes and lysosomes (Piper & Luzio, 2004, Wong, Li et al., 2012) and is permeant to multiple ions including, Ca²⁺, Fe²⁺, Na⁺, K⁺ and Zn²⁺(Cuajungco, Basilio et al., 2014, Dong, Cheng et al., 2008, LaPlante, Falardeau et al., 2002). Functionally, Ca²⁺ signalling mediated by TRPML1 has been linked to the biogenesis of lysosomes and the clearance of autophagic vacuoles via activation of the transcription factor EB (TFEB) (Medina et al., 2015, Medina, Fraldi et al., 2011).

Abnormal lysosomal Ca²⁺ signalling as a contributing factor to neurodegeneration and cellular dysfunction in the lysosomal storage diseases

Defects in lysosomal Ca²⁺ maintenance or signalling have been associated with human diseases, mainly with the lysosomal storage disorders (Lloyd-Evans et al., 2010), a group of predominantly childhood neurodegenerative diseases (Cox & Cachon-Gonzalez, 2012). Lysosomal storage diseases (LSDs), whilst being individually rare, are collectively the most common form of childhood neurodegenerative disease occurring at a combined frequency of 1:5,000 live births and comprise a family of >60 diseases (Cox & Cachon-Gonzalez, 2012). The majority are caused by defects in lysosomal enzymes, but an increasing cohort of ~20 are caused by genetic mutations in genes encoding for lysosomal transmembrane proteins (Lloyd-Evans & Platt, 2010). For almost all of these diseases the exact mechanisms that lead to neuronal cell death remain largely unknown and treatment options for most of the diseases are limited to palliative care(Platt & Jeyakumar, 2008). Work by us and others has led to the emergence of evidence connecting several of these disorders with defects in intracellular Ca²⁺ handling, that in some cases results in excitotoxicity, and has been directly shown to result in neuronal cell loss (Lloyd-Evans et al., 2008, Lloyd-Evans, Pelled et al., 2003a, Pelled, Lloyd-Evans et al., 2003, Pereira, Gazarini et al., 2010, Tessitore, del et al., 2004). The evidence for involvement of Ca²⁺ in the pathophysiology of these diseases is summarised in Table 1 and some of this work has been reviewed previously(Lloyd-Evans et al., 2010). In some cases the main defect has been alterations in Ca²⁺ handling in intracellular stores other than the lysosome (Korkotian, Schwarz et al., 1999), these diseases are not discussed in detail here but information can be found elsewhere (Vitner, Platt et al., 2010). Furthermore, new lysosomal diseases with defects in lysosomal Ca2+ homeostasis are being identified. One example being CLN3 disease, a member of the neuronal ceroid lipofuscinoses, caused by mutations in the CLN3 gene encoding the predominantly lysosomal CLN3 protein of unknown function(Chandrachud, Walker et al., 2015). Cells and tissues from this disease accumulate autofluorescent lipofuscin in lysosomes and have substantial abnormalities in the production and clearance of autophagic vacuoles(Cao, Espinola et al., 2006). A recent study has demonstrated the presence of elevated lysosomal Ca²⁺ in a cerebellar cell line from the CLN3 exon 7/8 1kb deletion mouse model(Chandrachud et al., 2015), the first report of such an increase in any disease. Further work is required to determine the cause of this defect and the role it may play in the pathogenesis of CLN3 disease.

Table 1. Intracellular Ca²⁺ defects associated with the lysosomal storage diseases.

Disease	Disease cause and phenotypes	Description of Ca2+ defect	Cause of the Ca2+ defect	Functional consequence of disrupted Ca2+
Lysosomal Ca2+ defects
Niemann-pick type C	Mutations in the NPC1 gene encoding a lysosomal transmembrane protein beleived to be invovled in lipid transport. Patient cells accumulate multiple lipids within lysosomes including cholesterol, sphingomyelin, sphingosine and glycosphlngoliplds	Reduced intravesicular levels resulting in reduced Ca2+ release via TPC2 and potentially TRPML1	Lysosomal accumulation of sphingosine	Disrupted endocytisis and lysosomal lipid storage, pharmacological elevation of Ca2+ rescues Purkinje neuron death in the mouse model
Mucolipidosis type IV	Mutations in the MCOLN1 gene encoding a late endosomal and lysosomal ion channel permeant to Ca2+, Fe2+, Na+, K+ and Zn2+. Patient cells are characterised by intralysosomal accumulation of phospholipids, sphingoids. mucopolysaccharides and free Fe2+	Potential eleveation in lysosomal Ca2+ content	Loss of function in late endosomal/lysosomal Ca2+ channel TRPML1	Defects in clearance of autophagic vacuoles within neurons
CLN3 disease	Mutations in the CLN3 gene encoding a lysosomal transmembrane protein, CLN3, of unknown function. cells accumulate autofluoreseent lipofuscin	Elevated lysosomal Ca2+	Currently unknown	Dirupted clearance of autophagic vacuoles
Mucopolysaccharidosis type I	Mutation of the IDUA gene encoding a-L-iduronidase which is a lysosomal enzyme that cleaves glycosaminoglycans, which accumulate in MPS type I	Elevated lysosomal Ca2+	Attributed to lysosomal deacidification	Elevated levels of apoptosis
ER Ca2+ effects
Gaucher disease	Mutation in GBA1 encoding acid b-glucosidase orglutosylceramidase. Cells accumulate the enzyme substrate, the glycosphlngolipld glueosyfceramlde within lysosomes	Enhanced Ca2+ release from the ER via potentiation of ryanodine receptors	Accumulating glucosylceramide, the lysosomal storage substance in Gaucher disease affacting ER function	Neuronal excitotoxicity and death as a result of elevated cytosolic Ca2+
GM1 gangliosidosis	Mutation in the GLB1 gene encoding lysosomal b-gaiactosidase. Cells accumulate the enzyme substarte ganglioside GM1, a important neuronal glycosphlngolipld, in lysosomes	Inhibition of ER Ca2+ entry via SERCA, elevated mitochondrial Ca2+	Accumulating ganglioside GMl, the Disrupted mitochondrial function. ER stress substrate of the lysosomal b-gafactosWase and presence of the unfolded protein enzyme defective in GMl gangliosidosis, response, altering ER function	Disrupted mitochondrial function, ER stress and presence of the unfolded protein response
GM2 gangliosidosis (SancShoff disease)	Mutation in HEXB gene encoding lysosomal b-hexosaminidase which degrades the intermediate ganglioside GM2 in lysosomes and which accumulates in Sandhoff disease	Inhibition of ER Ca2+ entry via SERCA	Accumulating ganglioside GM2, the substrate of the lysosomal b-hexosaminidase entyme detective in GM2 gangliosidosis, altering ER function	Enahnced sensitivty of primary neurons to excitotoxkity
Niemann-Pick disease type A	Mutation in the SMPD1 gene encoding the lysosomal enzyme acid sphingomyelinase, responsible for the degradation of the phospho-sphingolipld sphingomyelin within lysosomes	Inhibition of plasma membrane and ER Ca2+ ATPase activity reuslting in elevated cytosolic Ca2+	The accumulating lipid substrate of acid sphingomyelinase, the enzyme defective in Niemann-Pick A, sphingomyelin is implicated but mechanism ol inhibition currently unknown	Elevated oxidate stress in neurons from mouse model of NPA and from a NPA patient
LSDs with other Ca2+ defects Krabbe disease
Fabry disease	Mutation in the GLA gene encoding for a-gsiactosidase A. Loss of function causes the enzyme substrate, gioboside, a glycosphlngolipld, to accumulate	Enhanced voltage gated Ca2+ currents and elevated Ca2+ entry into sensory neurons	Lyso-globoside, a storage molecule in Fabry disease acts on plasma membrane voltage dependent Ca2+ channels	Potential cause of peripheral neuropathy
Pompe disease	Mutation in the GAA gene enoding a-glucosidase which breaks down glycogen within lysosomes	Mitochondrial Ca2+ overload and up-regulation of the Blsubunit of the L-type Ca2+ channel	Potential premature ageing of cells	Compromised mitochondrial respiration in whole muscle fibers
LSDS without Ca2+ defects
Cystinosis	Mutation in the CTNs gene that encodes cystinosina lysosomal transmembrane protein involved in the efflux of L-cystine from lysosomes	No lysosomal Ca2+	N/A	N/A

Notes: Niemann-Pick C, mucolipidosis type IV and CLN3 disease are reviewed in this article, mucopolysaccharidosis type I (Pereira et al., 2010), the lysosomal diseases with associated ER Ca²⁺ signalling defects (Ginzburg and Futerman, 2005; Korkotian et al., 1999; Pelled et al., 2003; Tessitore et al., 2004), Krabbe (Im, et al., 2001), Fabry (Choi, et al., 2015), Pompe (Lim, et al., 2015), and cystinosis (Ivanova, et al., 2016) are discussed elsewhere as indicated.

Niemann-Pick disease type C, a disease of reduced lysosomal Ca²⁺ and defective lipid endocytosis

The first reported case of a defect in lysosomal Ca²⁺ in a human disease was that of Niemann-Pick type C (NPC) disease (Lloyd-Evans et al., 2008). NPC is an LSD that manifests primarily with loss of cerebellar Purkinje neurons that results in ataxia, however, neuronal dysfunction is widespread and hallmarks of Alzheimer’s have also been reported (Lloyd-Evans & Platt, 2010). Patients usually succumb to the disease in their second decade of life but adult onset forms of the disease as well as milder disease causing mutations have all been reported(Millat, Marcais et al., 2001, Vanier & Millat, 2003). The disease is caused by mutation in the NPC1 gene that encodes a 13 transmembrane domain protein of the limiting membrane of the lysosome, also called NPC1 (Lloyd-Evans & Platt, 2010). The exact function of NPC1 remains to be identified but it is believed to be involved in lipid transport, especially cholesterol and potentially sphingolipids and fatty acids (Ioannou, 2005, Lloyd-Evans & Platt, 2010). NPC1 belongs to the eukaryotic family of resistance nodulation division (RND) permease multi-substrate pumps that are capable of transporting antibiotics, heavy metals, detergents, dyes and lipids, all of which have been reported to accumulate in NPC disease lysosomes (Davies, Chen et al., 2000, Kaufmann & Krise, 2008, Tseng, Gratwick et al., 1999). NPC lysosomes accumulate cholesterol, simple sphingolipids including sphingosine, glycosphingolipids, sphingomyelin and lyso-(bis)phospatidic acid (LBPA)(Lloyd-Evans et al., 2008, Lloyd-Evans & Platt, 2010). In 2008 we determined that multiple cell lines from NPC disease (NPC1 null Chinese hamster ovary cells, NPC1 null mouse astrocytes, NPC patient fibroblasts) as well as cells treated with the NPC1 protein inhibitor U18666a (Lu, Liang et al., 2015) all had reduced levels of lysosomal Ca²⁺ (measured both directly with intra-lumenal dextran conjugated Ca²⁺ probes as well as indirect pharmacological release of Ca²⁺ from lysosomes to the cytosol for measurement with a membrane permeant fluorescent Ca²⁺ dye) as well as reduced lysosomal Ca²⁺ release in response to membrane permeant NAADP-AM (Lloyd-Evans et al., 2008). The impact of this defect in lysosomal Ca²⁺ homeostasis in NPC disease is an inhibition of fusion and endocytic transport between the component parts of the endo-lysosomal system, leading to the large accumulation of lipids within NPC cells and tissues(Lloyd-Evans et al., 2008, Visentin, De Nuccio et al., 2013). Indeed the importance of the Ca²⁺ defect to disease pathology was highlighted by the use of curcumin as a potential therapy(Borbon, Hillman et al., 2012, Efthymiou, Steiner et al., 2015, Lloyd-Evans et al., 2008). Curcumin acts to elevate cytosolic Ca²⁺ levels by weak inhibition of SERCA and uncovering of the activity of the ER Ca²⁺ leak channels (Bilmen, Khan et al., 2001). Curcumin led to a significant improvement in function and lifespan of the NPC mouse model (Lloyd-Evans et al., 2008), with a significant rescue of Purkinje cell degeneration in the cerebellum(Smith, Wallom et al., 2009). However any benefit may well have been ameliorated by the action of curcumin against the cytochrome p450 enzymes (Wang, Sun et al., 2015), the activity of which have been shown to be considerably reduced in NPC disease (Nicoli, Al Eisa et al., 2016). The potential benefit of curcumin (in a lipidated vector) has also been questioned (Borbon et al., 2012) as has the cellular benefit (Yu, Swaroop et al., 2014), the low concentrations of curcumin used in this cell based study (2-5μM) are highly unlikely to induce significant elevation in cytosolic Ca²⁺ in order to rescue endo-lysosomal function in NPC disease cells. Indeed, it is this requirement for higher concentrations of curcumin (10-30μM (Lloyd-Evans et al., 2008)), higher than those found in the plasma and CSF (0.2-1.8μM (Mishra & Palanivelu, 2008)), to induce Ca²⁺ elevation within cells that has led to the speculation that curcumin cannot mediate benefit in this way in NPC disease (Borbon et al., 2012). However, it should be noted that it’s effects are not due to its well-known antioxidant properties (Smith et al., 2009) and low doses of curcumin given to Tg2576 mutant APP overexpressing Alzheimer mice over a period of time were capable of crossing the blood brain barrier and accumulating in the CNS where they stained amyloid deposits(Yang, Lim et al., 2005). As such, it is possible that curcumin eventually reaches a high enough concentration within cells to weakly inhibit SERCA and induce changes in Ca²⁺, further work is required in this area.

The defect in lysosomal Ca²⁺ in NPC disease was shown to be triggered by the accumulation of sphingosine (Lloyd-Evans et al., 2008), which has recently been suggested to activate TPC1 and induce emptying of the lysosomal Ca²⁺ store (Hoglinger, Haberkant et al., 2015). This is an interesting hypothesis and one that requires testing by inhibiting the Ca²⁺ release with the NAADP receptor inhibitor Ned19 (Naylor, Arredouani et al., 2009), which should therefore theoretically rescue NPC disease phenotypes. It is also interesting to note that the accumulation of sphingosine in NPC disease cells (~1μM (Lloyd-Evans & Platt, 2010)) is not sufficient to alter lysosomal pH (which is normal in NPC disease (Bach, Chen et al., 1999, Lloyd-Evans et al., 2008)), or induce lysosomal membrane rupture, which can happen at higher concentrations of sphingosine (Kagedal, Zhao et al., 2001, Lloyd-Evans & Platt, 2011). As it has been reported that activation of Ca²⁺ release from lysosomes with NAADP induces a change in lysosomal pH (Morgan & Galione, 2007), it would appear that this enhanced Ca²⁺ release is not occurring constitutively, even though lysosomal Ca²⁺ levels remain constitutively lower (Lloyd-Evans et al., 2008). Furthermore, it has been shown that it requires ~10μM sphingosine or more to induce Ca²⁺ release from lysosomes (Lloyd-Evans & Platt, 2011), a concentration ~10 fold greater than is actually found in NPC disease storage lysosomes (Lloyd-Evans & Platt, 2010). Another possibility is that sphingosine accumulation is inhibiting the mechanism of Ca²⁺ entry into NPC disease lysosomes, as it is a known inhibitor of Ca²⁺ ATPases(Colina, Cervino et al., 2002, Pandol, Schoeffield-Payne et al., 1994), the activity of which have been found on purified lysosomes(Ezaki, Himeno et al., 1992). Clearly more work is required to establish the exact mechanism by which lysosomal Ca²⁺ levels are reduced in NPC disease.

One study has suggested that lysosomal Ca²⁺ levels are in fact not reduced in NPC disease (Shen, Wang et al., 2012). This study is in contention with multiple reports from various research labs (Coen et al., 2012, Hoglinger et al., 2015, Lee, Lee et al., 2010a, Lee, Lee et al., 2014, Lloyd-Evans et al., 2008, Speak, Te Vruchte et al., 2014, Visentin et al., 2013, Xu, Liu et al., 2012). Several of these labs have used either the same NPC patient fibroblast cell line (Visentin et al., 2013, Xu et al., 2012) that was used in the original study(Lloyd-Evans et al., 2008), or a U18666a induced cellular model of NPC disease(Coen et al., 2012, Lloyd-Evans et al., 2008), and all of whom have reported a reduction in lysosomal Ca²⁺ in NPC disease and often by various methods in multiple cells including Purkinje neurons(Lee et al., 2010a). One possibility for the discrepancy in the results is indeed the tools and methods used in the studies. In our initial paper we presented data that NPC disease B lymphoblasts had lower lysosomal Ca²⁺ than controls using the lysosomal cathepsin C substrate GPN alone to burst lysosomes(Lloyd-Evans et al., 2008). The data from the other NPC cell lines used in the paper (CHO, human patient fibroblast and RAW macrophages treated with the NPC1 inhibitor U18666a) was the same but we had to use a slightly different method to fully realise the difference in lysosomal Ca²⁺ that we had also measured with an in situ dextran conjugated Ca²⁺ probe(Lloyd-Evans et al., 2008). In these other cell lines we first clamped the other intracellular stores with either thapsigargin (to release ER Ca²⁺) or ionomycin (to release all intracellular Ca²⁺ stores apart from lysosomes) prior to releasing lysosomal Ca²⁺ with GPN(Lloyd-Evans et al., 2008). We did not require any clamping of the other intracellular Ca²⁺ stores in the B lymphoblasts as the mechanisms governing potentiation of lysosomal Ca²⁺ release by the ER appear to be different in these immortalised B cells (Waller-Evans and Lloyd-Evans unpublished, (Dellis, Arbabian et al., 2009)). Shown in Fig. 2 is the difference we observe in lysosomal Ca²⁺ content in fibroblasts when we compare cells that have had the intracellular stores clamped prior to either NAADP-AM or GPN addition versus those that have not. As can be seen, there is a substantially smaller lysosomal Ca²⁺ release in the cells which have not been clamped, which correlates more closely with the intra-lumenal measurements and the low volumetric content of lysosomes within cells. It is therefore clear that clamping of intracellular stores is crucial in order to conduct accurate indirect measurements of the content of the lysosomal Ca²⁺ store, it is unclear whether this is the case in Shen et al.

Figure 2. Lysosomes induce Ca²⁺ release from the ER.

Utilisation of either GPN to burst lysosomes to measure Ca²⁺ levels or NAADP-AM to release Ca²⁺ from lysosomes results in a large amount of Ca²⁺ release (Fig Ai and Aii). This is the result of lysosomal Ca²⁺ release inducing further Ca²⁺ release from the ER (Fig. Bi) which can mask real changes in lysosomal Ca²⁺ levels in cells. In order to properly quantify lysosomal Ca²⁺ it is necessary to clamp the Ca²⁺ release from the other stores, this can be done by pretreament of the cells with an inhibitor of Ca²⁺mobilisation such as thapsigargin which blocks Ca²⁺ uptake into the ER via inhibition of SERCA that subsequently uncovers Ca²⁺ leak out of the ER (Ai and Aii). Following release of the ER store, GPN or NAADP-AM can be added to give a more accurate, smaller, estimate of lysosomal Ca²⁺, this is summarised in Bii.

Is there a role for TRPML1, the lysosomal Ca²⁺ channel defective in mucolipidosis type IV, in the pathogenesis of Niemann-Pick disease type C?

Inhibition of TRPML1 by lysosomal sphingomyelin has been suggested as a possible mechanism for altering lysosomal Ca²⁺ homeostasis in NPC disease(Shen et al., 2012). That sphingomyelin is unlikely to affect TRPML1 is supported by the findings that inhibition of sphingolipid biosynthesis with myriocin, which reduces sphingosine levels first, rescues the lysosomal Ca²⁺ defect prior to any correction in sphingomyelin levels(Lloyd-Evans et al., 2008). Furthermore, the authors indicate that overexpression of TRPML1 or its activation with the agonist MLSA1 is capable of correcting the defects in endocytic trafficking and cholesterol accumulation observed in different NPC cell lines(Shen et al., 2012). It is therefore interesting to point out that some of their lysosomal Ca²⁺ measurements in NPC cells were made using a construct that overexpresses a Ca²⁺ measuring GCaMP attached to TRPML1(Shen et al., 2012), which in itself may not distinguish between Ca²⁺ release from lysosomes or more global Ca²⁺ signalling events as highlighted by(Kilpatrick, Yates et al., 2016b). If overexpression of TRPML1 rescues NPC lysosomal phenotypes then theoretically this may correct the defect in lysosomal Ca²⁺ so that there is ultimately no difference to measure. It is also interesting to note that in a separate study by the same group there was no effect of MLSA1 on lysosomal cholesterol in NPC1 null Chinese hamster ovary cells, NPC1 null macrophages or U18666a treated control macrophages(Wang, Gao et al., 2015). This discrepancy is yet to be fully explained. Finally, should TRPML1 be rendered dysfunctional in NPC disease then one would expect some degree of phenotypic overlap between NPC disease cells and cells from the lysosomal disease mucolipidosis type IV (MLIV) where genetic mutations in the MCOLN1 gene render the gene product, TRPML1, inactive, dysfunctional or absent(Bach, 2001).

Does pathogenesis of mucolipidosis type IV mirror Niemann-Pick disease type C?

We have previously summarised the main differences in phenotypes at the cellular level between NPC and MLIV (Lloyd-Evans & Platt, 2011) we now summarise the main differences in patient symptoms (table 2). Although the two diseases are clearly genetically distinct, one would expect some significant degree of overlap in phenotypes if TRPML1 was rendered dysfunctional in both diseases. For example, autofluorescence is well recorded in MLIV cells and tissues but reports of lipofuscin accumulation in NPC disease do not exist in the literature apart from one study (Shen et al., 2012) where apparent autofluorescent material in NPC cells is co-localised against lysotracker red, a probe well known for its ability to rapidly photoconvert into a green fluorescent molecule (Freundt, Czapiga et al., 2007). At the patient level, a major phenotype in MLIV is elevated blood gastrin levels, iron deficiency in some patients and the presence of achlorhydria from defects in Parietal cell function which affects the acidity of gastric secretions(Schiffmann, Dwyer et al., 1998). There have been no achlorhydria in NPC patients. Ultimately, based on the weight of evidence from a number of different research groups, it would appear that lysosomal Ca²⁺ is reduced in NPC disease. Indeed, this reduced lysosomal Ca²⁺ level defect has been used as a phenotypic output in a high throughput drug screen in NPC patient fibroblasts by the NIH (Xu et al., 2012) and elevation of lysosomal Ca²⁺ has been shown to be beneficial in a number of independent studies (Visentin et al., 2013, Xu et al., 2012).

Table 2. Clinical differences and similarities between Niemann-Pick type C and mucolipidosis type IV patients.

	Mucolipidosis type IV	Niemann-Pick type C
Neurological symptoms
Cerebellar neuron loss	Present late in disease progression	Early event in pathology
Thinned corpus callosum	Present	Not Present
Developmental delay	Severe	Present
Cognitive impairment	Present	Present
Epilepsy	Not present	Present
Cataplexy	Not present	Present
Dystonia	Not present	Present
Tremor	Not present	Present
Psychiatric disorders	Not present	Present
Dementia	Not present	Present
Hearing loss	Not present	Present
Opthalmic symptoms
Ocular-motor abnormalities	Not present	Present
Corneal clouding	Present	Not present
Retinal degeneration	Present	Not present
Strabismus	Present	Not present
Visceral symptoms
Hepatospenomegaly	Not present	Present
Cholestasis	Not present	Present
Dysphagia	Present	Present
Dysarthria	Present	Present
Achlorydia	Present	Not present
Elevated gastrin	Present	Not present
Poor physical growth	Present	Not present
Hypotonia	Present	Present
Lung disease	Not present	Present
Disease progression
Progressive decline	Not present	Present
Stability after initial decline	Present	Not present

Notes: Overall, only 4/26 phenotypes are shared and these are features that are aligned amongst most neurodegenerative diseases (Altarescu, Sun et al., 2002; Patterson, et al., 2012; Patterson, et al., 2013; Schiffmann et al., 1998; 2014; Smith, et al., 2002). It appears there is little similarity between NPC and MLIV at the patient level, as is the case at the cellular level (Lloyd-Evans and Platt, Cell Calcium 2011).

What is the role of loss of TRPML1 function in the pathogenesis of mucolipidosis type IV?

Loss of function of TRPML1 in MLIV disease results in a clinical phenotype that is embodied by initial rapid neurological decline during early infancy followed by stabilisation for multiple decades(Bach, 2001). The disease is also characterised by ophthalmological defects, abnormal blood gastrin levels and achlorhydria(Schiffmann et al., 1998). At the cellular level, there is an accumulation of lipids (gangliosides and phospholipids(Bach, Zeigler et al., 1980, Bargal & Bach, 1988, Bargal & Bach, 1989)), glycosaminoglycans (Bach, Ziegler et al., 1977) and Fe²⁺ (Dong et al., 2008) within lysosomes that themselves have altered pH and accelerated lipid recycling(Bach et al., 1999, Chen, Bach et al., 1998). Mutations in the MCOLN1 gene, that encodes TRPML1, are the cause of MLIV(Bargal, Avidan et al., 2000). As mentioned earlier, TRPML1 is a late endosomal and lysosomal ion channel permeant to multiple ions including, Ca²⁺, H⁺, Na⁺, K⁺ and Zn²⁺(Cuajungco et al., 2014, Dong et al., 2008, LaPlante et al., 2002). Interestingly, recent work has indicated that TRPML1 may be involved in triggering Ca²⁺ release from the ER (Kilpatrick et al., 2016b) suggesting an impact on global Ca²⁺ signalling as well as an ability to regulate autophagy through activation of TFEB via the Ca²⁺ dependent calcineurin(Medina et al., 2015). It has also been reported that TRPML1 is regulated by pH(Cantiello, Montalbetti et al., 2005, Raychowdhury, Gonzalez-Perrett et al., 2004), a finding which was unexpectedly supported in familial Alzheimer’s disease where loss of function mutations in PSEN1 encoding presenilin 1 (PSEN1) leads to defects in lysosomal pH that activate TRPML1 and empty the lysosomal Ca²⁺ store (discussed in more detail below) (Lee et al., 2015). The vast majority of work on TRPML1 has been done using constitutively active mutant forms of TRPML1, how exactly these mutant proteins recapitulate the function of wild-type TRPML1 remains largely unknown(Waller-Evans & Lloyd-Evans, 2015).

Common neurodegenerative diseases with defects in lysosomal Ca²⁺

Parkinson’s disease

Parkinson’s disease (PD), one of the most common neurodegenerative diseases, is a progressive idiopathic neurodegenerative disease of ageing that affects >1% of the population aged over 65 and >4% of over 85’s (de Rijk, Launer et al., 2000). As well as age, both genetic (PARK gene family) and environmental factors (e.g. traumatic brain injuries, exposure to pesticides, exposure to metals such as Mn²⁺) have been identified as contributors to the prevalence of this disease(Lai, Marion et al., 2002). PD is the most common movement disorder(Tong, Yamaguchi et al., 2010), the motor defects synonymous with PD are caused by loss of the dopaminergic neurons in the substantia nigra and dopaminergic terminals in the striatum(Dauer & Przedborski, 2003). Loss of neurons is associated with the presence of intracellular inclusions known as Lewy bodies with low molecular weight oligomers of α-synuclein thought to be the primary cause of the neurotoxicity(Planchard, Exley et al., 2014). Evidence that Ca²⁺ signalling is critical in PD for survival of the dopaminergic neurons in the substantia nigra pars compacta comes from the observation of reduced susceptibility to degeneration in neurons expressing the Ca²⁺ binding and buffering protein calbindin-D28K(Yamada, McGeer et al., 1990). Although several studies exist on the role of changes in Ca²⁺ signalling in inducing neuronal excitotoxicity in PD(Schulz, 2007), changes in lysosomal Ca²⁺ signalling have only been explored in recent years. Neuronal cell death in PD is associated to the release of ATP from the cytosol to the extracellular space. This elevation, comparing 1-10nM ATP in the extracellular space to 10mM in the cytosol, results in both an increase in α-synuclein and the activation of ATP responsive P2X channels on neighbouring neurons and the influx of Ca²⁺ into the cell. Interestingly, this elevation in Ca²⁺ mediated by P2X channels was shown to induce lysosomal alkalinisation and an increase in autophagic vacuoles(Gan, Moussaud et al., 2015). Further evidence for a potential role of lysosomal Ca²⁺ in PD emerged from a pharmacologically induced model where cathepsin B and L activity, known to be important in lysosomal α-synuclein degradation(McGlinchey & Lee, 2015), was inhibited by Z-Phe-Ala-diazomethylketone. Neurons treated with this inhibitor had expanded lysosomes, elevated lysosomal Ca²⁺ and elevated lysosomal Ca²⁺ release in response to NAADP-AM(Dickinson, Churchill et al., 2010). Similar phenotypes (described below) have now been identified in various PD models.

A subset of the PARK genes are associated with lysosomes and the endo-lysosomal system, these include; the lysosomal p-type ATPase 13A2 (PARK13), the leucine-rich repeat kinase LRRK2 (PARK8) and the lysosomal enzyme glucocerebrosidase (GBA1). The GBA1 gene has recently emerged as a major risk factor for PD with an estimated 10% of sporadic cases of PD thought to be caused by heterozygous mutations in this gene(Sidransky, Nalls et al., 2009). Whilst the exact mechanisms linking reduced activity of glucocerebrosidase (GlcCerase) to loss of substantia nigra dopaminergic neurons remain unknown there has been compelling evidence linking mis-folded GlcCerase to ER dysfunction. The accumulation of mutant GlcCerase in the ER leads to elevated stress (Westbroek, Gustafson et al., 2011) and abnormally elevated ER Ca²⁺ release has been reported in PD patient fibroblasts carrying the N370S mutation in GBA1(Kilpatrick et al., 2016a). Indeed, abnormal ER Ca²⁺ release via the ryanodine receptor is a hallmark of Gaucher disease (Korkotian et al., 1999, Pelled, Trajkovic-Bodennec et al., 2005), the most common lysosomal disease caused by loss of function mutations in GBA1, and is observed in patient post-mortem brain as well as in mouse neurons where GlcCerase is pharmacologically inhibited by conduritol β-epoxide (CBE)(Korkotian et al., 1999, Pelled et al., 2005). In PD caused by heterozygous mutations in GBA1 the cause of the ER Ca²⁺ defect is likely different to Gaucher disease as it is unlikely that there is any storage of the GlcCerase substrate glucosylceramide, as residual enzyme activity must drop to below 15% of normal for lysosomal glucosylceramide storage to occur as is observed in Gaucher disease(Schueler, Kolter et al., 2004). Further evidence that the ER Ca²⁺ defect in PD does not occur as a result of lipid storage is that this phenotype is observed in PD patient fibroblasts, which are known to not accumulate glucosylceramide(Westbroek, Nguyen et al., 2016). It therefore appears that there is a significant difference between PD caused by heterozygosity in GBA1 and Gaucher disease as although both diseases have abnormal ER Ca²⁺ homeostasis the underlying cause is different as it is the lipid storage that induces this phenotype in Gaucher disease(Lloyd-Evans et al., 2003a, Lloyd-Evans, Pelled et al., 2003b). It is interesting however to note that a defect in lysosomal Ca²⁺ levels was observed in PD patient fibroblasts carrying the N370S mutation, with lower Ca²⁺ levels being released into the cytosol of the cells following osmotic lysis of lysosomes with GPN(Kilpatrick et al., 2016a). This phenotype appears to resemble NPC disease where the accumulation of Lewy bodies in NPC patient post-mortem brain has been reported (Chiba, Komori et al., 2014), suggesting a potential connection. It is also interesting to note that the activity of GlcCerase is reduced in NPC cells as a result of mis-localisation of the enzyme to late endosomes(Salvioli, Scarpa et al., 2004), alongside the accumulation of cholesterol in both NPC disease and Gaucher disease(Lloyd-Evans & Platt, 2010, Ron & Horowitz, 2008), there are some compelling simialrities that warrant further investigation in future. Interestingly, no ER Ca²⁺ defect was observed in fibroblasts where GlcCerase was inhibited by CBE (only in the PD patient fibroblasts heterozygous for GBA1 mutations, (Kilpatrick et al., 2016a)), which contrasts with earlier studies on Gaucher disease (Korkotian et al., 1999). However it should be noted that although GlcCerase activity was reported as being negligible in these CBE treated cells, the concentration of CBE used (10μM) was low and based on other studies would potentially inhibit GlcCerase to levels no lower than at least 25% of control (Dermentzaki, Dimitriou et al., 2013), which, based on 15% of enzyme activity being enough to prevent lysosomal storage(Schueler et al., 2004), would not lead to a phenotype resembling Gaucher disease (which would not happen in fibroblasts either as they do not store glucosylceramide) but would in fact be more similar to the level of residual enzyme activity in PD. The very fact that this result with the GlcCerase inhibitor CBE does not resemble what is seen in PD patient cells where GlcCerase is potentially mis-folded again indicates that the mechanisms underlying the Ca²⁺ defects in both diseases are quite different, even if they do ultimately resemble one another.

Autosomal dominant mutations in PARK8 encoding LRRK2 is one of the most common genetic causes of PD and result in a rare late-onset familial PD that is indistinguishable from sporadic forms of PD (Gomez-Suaga, Luzon-Toro et al., 2012, Tong et al., 2010). Large genome wide association studies (GWAS) have also shown the presence of more common genetic variants in LRRK2 associated with the risk of non-familial PD(Simon-Sanchez, Schulte et al., 2009). Whilst the exact function of the LRRK2 protein is unknown it has been shown to have both Ras-like GTPase and MAPKKK-like kinase domains (Marin, 2006) and is a protein that is found on the ER as well as endo-lysosomes(Biskup, Moore et al., 2006). A role for LRRK2 in endo-lysosomal function first emerged from the findings that mice null for LRRK2 present with elevated levels of α-synuclein, elevated levels of autophagy, as indicated by an increase in the autophagosomal marker LC3-II, and an increase in lipofuscin, indicative of lysosomal dysfunction (Tong et al., 2010). These findings have been further supported from research into invertebrate models of PD, the Drosophila homologue of LRRK2 (Lrrk) localises exclusively to late endosomes and lysosomes where it interacts with and negatively regulates the function of Rab7, a GTPase that regulates endocytic transport. Wild-type LrrK appears to restrict the perinuclear localisation of lysosomes, whilst a Drosophila mutant lrrk that resembles the pathogenic human PD causing LRRK2^G2019S mutation promotes perinuclear lysosomal clustering with the presence of occasionally expanded late endosomes (Dodson, Zhang et al., 2012). Interestingly, expression in mouse primary astrocytes of LRRK2 harbouring not only the common LRRK2 mutation G2019S but also the expression of other mutations such as R1441C or Y1699C all led to enlarged lysosomes, a phenotype that could be reversed using a LRRK2 kinase inhibitor(Henry, Aghamohammadzadeh et al., 2015). Of interest to the potential role of LRRK2 in modulating endosome and lysosome function in the pathophysiology of PD are the findings from genetic studies of PD patient cohorts indicating increased risk of PD caused by overlap of common variants in LRRK2 with variants at another PD risk associated locus, PARK16 (MacLeod, Rhinn et al., 2013, Pihlstrom, Rengmark et al., 2015). Although several genes are contained within this locus, one in particular, namely RAB7-like variant 1 (RAB7L1), provides a further connection to dysfunctional endo-lysosomes in PD and a genetic link between LRRK2 mutations and altered function of endocytic Rab7 GTPases. Furthermore, inhibition of Rab7 in PD patient fibroblasts carrying the common G2019S mutation in LRRK2 led to a correction in the perinuclear clustering of lysosomes (Hockey, Kilpatrick et al., 2015), providing further evidence of the interplay between LRRK2 and Rab7 in regulating endo-lysosomal function in both healthy cells (negative regulation) and in PD (gain of function). Interestingly, the beneficial effect of inhibiting Rab7 function on lysosomal clustering in PD patient cells could be replicated by genetic silencing of the expression of the lysosomal Ca²⁺ channel TPC2 (but not the endosomal channel TPC1) or inhibition of the TPC2 channel with Ned-19. This benefit was attributed to the presence of a defect in lysosomal Ca²⁺ in the PD patient cells with greater NAADP mediated Ca²⁺ release from lysosomes observed in the PD cells compared to controls(Hockey et al., 2015). Over-expression of TPC2, and elevated lysosomal Ca²⁺ release, has been connected with defects in endocytosis and autophagic vacuole clearance with an associated defect in recruitment of Rab7 to autophagosomes(Lu, Hao et al., 2013). Furthermore, TPC2 has been shown to co-immunoprecipitate with LRRK2 (Gomez-Suaga et al., 2012) raising the possibility that mutant LRRK2 may lead to enhanced activity of TPC2 and greater lysosomal Ca²⁺ release via abnormal TPC2 phosphorylation resulting in lysosomal accumulation and defects in autophagic vacuole clearance via altered Rab7 recruitment. This raises the possibility that treating downstream abnormal lysosomal Ca²⁺ signalling may be a potential therapeutic strategy for certain forms of PD, especially as inhibition of TPC2 with Ned-19 has been shown to rescue the defects in lysosomal clustering (Hockey et al., 2015) and the accumulation of autophagic vacuoles (Gomez-Suaga et al., 2012) associated with abnormal LRRK2 function.

Indeed, the possibility that modulating lysosomal Ca²⁺ is a therapeutic strategy that could be looked at more broadly for PD is supported by the recent study indicating that ambroxol, a drug that is used as an expectorant and is a known chaperone for GlcCerase(Ambrosi, Ghezzi et al., 2015), can induce Ca²⁺ release from lysosomes via alkalinisation(Fois, Hobi et al., 2015). However, it should be noted that the levels of LAMP1 and 2, LIMP2 and GlcCerase have all been reported to be lower in multiple regions of the PD brain, as well as the substantia nigra, (Chu, Dodiya et al., 2009, Murphy, Gysbers et al., 2014, Rothaug, Zunke et al., 2014). Although, whether or not these cases of PD were associated with mutations in LRRK2 is unclear but would suggest that further work is required and that the lysosomal Ca²⁺ phenotypes discussed above are not only disparate but also may not be a universal feature of PD.

Alzheimer’s disease

Alzheimer’s disease (AD) is a complex, multifactorial and polygenic disease of aging that constitutes a major healthcare burden worldwide. AD is a disease of the aging brain where the normal processing, breakdown and recycling of proteins such as the amyloid precursor protein in cells of the brain is deficient. How exactly these defects lead to dysfunction and loss of neurons remains to be fully elucidated but there is an emerging role of lysosomes. For a comprehensive background on the role of lysosomes in AD see(Lloyd-Evans & Haslett, 2016a). As with PD, lysosomal Ca²⁺ defects and an expanded lysosomal system have also been reported in AD(Wilson, Murphy et al., 2004). In particular, FAD caused by mutations in the PSEN1 gene encoding Presenilin 1 (PSEN1) a component of the γ-secretase complex that cleaves the amyloid precursor protein(Coen et al., 2012). Changes in lysosomal pH have been reported multiple times in PSEN1 null and mutant cells (Avrahami, Farfara et al., 2013, Coffey, Beckel et al., 2014, Lee et al., 2015, Lee, Yu et al., 2010b), suggesting a posibile cause of the altered lysosomal Ca²⁺ levels. It has been reported that the lysosomal pH defects in PSEN1 null cells are caused by abnormal glycosylation, in the absence of PSEN1, of the V0a1 subunit of the v-ATPase (Lee et al., 2015, Lee et al., 2010b). This in turn results in reduced v-ATPase activity and an alkalinisation of lysosomes which was proposed to cause defects in lysosomal protein processing and reduced clearance of autophagic vacuoles(Lee et al., 2015, Lee et al., 2010b). It has also been suggested that there is no defect in lysosomal pH in PSEN1 null cells and that the accumulation of autophagic vacuoles and lysosomal processing defects are the result of abnormal fusion between these compartments caused by the reduced lysosomal Ca²⁺(Coen et al., 2012). Interestingly, these findings suggest a possible mechanistic similarity to NPC disease, discussed above, especially as the NPC1 inhibitor U18666a (Lu et al., 2015) induces the same lysosomal Ca²⁺ dysfunction in all studies(Coen et al., 2012, Lee et al., 2015, Lloyd-Evans et al., 2008). Furthermore, NPC patients present with some hallmarks of Alzheimer like pathology(Saito, Suzuki et al., 2002). However, it appears that the mechanism governing lysosomal Ca²⁺ dysfunction in PSEN1 null cells and NPC disease cells is quite different. The change in lysosomal pH in PSEN1, which is not observed in NPC disease(Bach et al., 1999, Lloyd-Evans et al., 2008), triggers a number of changes in the mechanisms that govern lysosomal Ca²⁺ homeostasis. First, NAADP has been shown to be incapable of dissociating from purified TPC2 when luminal lysosomal pH is less acidic. This results in an inability of NAADP to trigger further Ca²⁺ release via TPC2 (Pitt et al., 2010). We have shown that NAADP-AM signalling is completely inhibited in PSEN1 null cells, providing supportive evidence for a change in lysosomal pH(Lee et al., 2015). This change in NAADP signalling was not caused by any alteration in TPC2 protein levels but could have been the result of overall reduced lysosomal Ca²⁺ levels in the PSEN1 cells. However, NAADP-AM induced no release from PSEN1 in comparison to U18666a treated cells, an inhibitor of lysosomal NPC1 which induced a similar reduction in lysosomal Ca²⁺ to that seen in PSEN1, where there was ~80% less Ca²⁺ release compared to control(Lee et al., 2015). This suggested to us that TPC2 had been rendered inactive by a change in lysosomal pH. As mentioned above, TRPML1 has been suggested to be activated following a change in lysosomal pH, potentially operating in late endosomes or homotypic organelles (Piper & Luzio, 2004, Raychowdhury et al., 2004). Based on the change in lysosomal pH in PSEN1 cells we tested this theory and found that the synthetic agonist of TRPML1, MLSA1, was able to induce significantly more Ca²⁺ release from PSEN1 lysosomes(Lee et al., 2015). We concluded that hyperactive TRPML1 was the cause of the alteration in lysosomal Ca²⁺ as inhibition of PIKfyve, the enzyme that creates PI(3,5)P₂ the endogenous ligand of TRPML1, was able to normalise PSEN1 lysosomal Ca²⁺(Lee et al., 2015). It is also interesting to note that the amyloid precursor protein (APP) itself binds to PIKfyve and regulates the levels of PI(3,5)P₂ (Currinn, Guscott et al., 2016)providing the possibility that APP itself regulates TRPML1 activity.

Interestingly, TRPML1 activity has also been shown to be altered in other forms of dementia. Amyloid-β accumulates by an unknown mechanism within neurons of brains infected with HIV and have been shown to accumulate in lysosomes laden with Ca²⁺ and sphingomyelin (Bae, Patel et al., 2014). Interestingly, activation of TRPML1 cleared both the amyloid accumulation as well as the accumulation of sphingomyelin in cultured neurons expressing the HIV coat protein gp120. Although this is the opposite of what we found in FAD, the cellular phenotypes in part resemble NPC disease, where there is also an accumulation of sphingomyelin. Furthermore, NPC1, sometimes referred to as a form of childhood dementia owing to the earliest recorded presence of neurofibrillary tangles, hyerphosphorylated tau and accumulation of amyloid-β_40-42 in post-mortem tissue samples from young patients (Saito et al., 2002, Suzuki, Parker et al., 1995), also has altered Ca²⁺ release from TRPML1(Shen et al., 2012). This is presumably in response to reduced late endosome/lysosomal Ca²⁺ levels rather than either a change in lysosomal pH (not present in NPC), or an effect of sphingomyelin on TRPML1 function as discussed above. Whether or not the reduced activity of TRPML1 in NPC disease is in any way involved with the cellular hallmarks of AD remains to be elucidated but there is clearly a role for altered lysosomal Ca²⁺ in certain forms of AD, demonstrating whether this feature also occurs in sporadic AD may be crucial for the development of any therapeutic strategy in this area.

To conclude, changes in lysosomal Ca²⁺ are becoming increasingly prevalent in various forms of both common and rare forms of neurodegenerative disease. The development of clinically approved therapeutics in the future that can target and alter lysosomal Ca²⁺ signalling may be an important avenue for ultimately treating these diseases.