Lysosomes are commonly viewed as the disposal system of the cell, but are recognized to have a secretory function in many cell types [484]. Lysosomes are secreted to the cell surface for
membrane repair [485,486]. Furthermore, in neurons lysosomes potentially provide the membrane required for neurite outgrowth [487]. They are involved in the secretion of thyroid hormone, pulmonary surfactant, albumin, and cytotoxic molecules from cytotoxic T lymphocytes (CTLs) [485,488,489]. Vesicles carrying these cargo belong to a family of lysosome-related organelles (LROs) [490]. LROs share some hydrolases and membrane proteins with lysosomes, but are unique in the cargo they carry to perform specialized roles.
The mechanism underlying LRO and conventional lysosome secretion is complex and requires the cooperation of many proteins. Before successful degranulation, each secretory granule must be delivered to the cell surface, tethered, docked, and stimulated to release by increased
intracellular calcium [491]. Genetic disruption of steps in LRO release can lead to familial hemophagocytic lymphohistiocytosis (FHL) or Griscelli syndrome (GS).
Griscelli syndrome is a autosomal recessive disease that is characterized by partial albinism and immunodeficiency [492]. There are three types of GS and they affect genes expressing a group of three interacting proteins (myosin Va, Rab27a, and melanophilin). Type 2 GS is caused by a mutation in Rab27a [493]. In GS, melanocytes accumulate pigment in melanosomes and natural killer (NK) cells have decreased cytotoxic activity [494]. Expression of dominant negative mutants of Rab27b (a brain expressed isoform) [495] in PC12 cells decreases ACTH secretion from dense core granules [496]. These deficits are likely due to disrupted LRO trafficking and plasma membrane docking. Rab27a deficient melanocytes missort melanosomes to the
perinuclear region [497]. Expression of dominant negative mutants of Rab27a in a melanocyte cell line phenocopies the melanosome distribution [498]. In addition to vesicle delivery to the subplasmalemmal region, Rab27 likely also participates in vesicle docking. Lytic granules in
CTLs from ashen mice (Rab27a KO mice) can polarize towards the target cell, but do not dock
at the plasma membrane [499]. Furthermore, knockdown of Rab27a or Rab27b leads to
increased mobility of lysosomes at the cell surface, which suggests that Rab27 may participate in lysosomes and LRO docking [500]. Therefore, Rab27 has an important role governing the
The function of Rab27 depends on the function of its many effectors. Rab27 effectors have an amino terminal Rab interacting domain and, typically, two carboxyl terminal C2 domains. The C2 domains are not present in all interacting proteins [501]. These effectors facilitate the docking and transport functions of Rab27 by facilitating interaction with microtubules, actin filaments, and SNARE complexes [501,502].
Melanophilin (Mlph) is a Rab27 interacting protein and mutations that impair its interaction with Rab27 result in GS type 3 [503]. In addition to Rab27, Mlph also interacts with the actin motor, Myosin Va (MyoVa), which results in a tripartite complex [504]. Mutation of any of these three proteins results in GS [505]. Indeed, expression of the Rab27 binding domain of Mlph acts as a dominant-negative, likely by sequestering active Rab27, causing melanosomes to accumulate in the perinuclear area [504]. Interestingly, Mlph also tracks the plus end of microtubules, through an interaction with the microtubule associated end binding protein 1 (EB1). This suggests that it may participate in melanosome delivery to cell periphery [506]. MyoVa also tracks the
microtubule plus end in a Mlph dependent manner [506]. However, in the absence of MyoVa, bidirectional microtubule transport of melanosomes is normal [507]. However, in the absence of MyoVa, melanosomes are not captured in the actin-rich periphery and become concentrated in the perinuclear region [508,509]. Therefore, the Rab27a, Mlph, and MyoVa complex is involved in vesicle trafficking to the cell periphery and efficiently dock them at the cell membrane. Another Rab27 effector, Exophilin 7 in neutrophils, is involved in the organization of actin filaments. Exophilin 7 interacts with the RhoA-GTPase-activating protein (GAP), Gem-
interacting protein (GMIP), and participates in actin filament deploymerization [510]. Cortical actin serves as a physical barrier between vesicles and the cell membrane [511,512]. At the cell periphery, azurophilic granules, from human neutrophils, can traverse cortical actin barrier by depolymerizing actin. However, preventing actin depolymerisation, by loss of GMIP-mediated RhoA inactivation, decreases granule attachment to the membrane [510].
In addition to trafficking and docking to the cell periphery, there is also cellular machinery to facilitate the fusion of lysosomes and LROs with the cell surface. The mechanism responsible is the SNARE machinery, which was first identified in studies of synaptic vesicle exocytosis. In
synaptic vesicle exocytosis, the formation of the SNARE complex is thought to provide the energy needed for membrane fusion. Target SNARE (t-SNARE) proteins on the plasma
membrane (SNAP-23, synaptobrevin 2) interact with vesicular SNARE (v-SNARE) proteins to form a four-helix bundle [513].
The exact SNARE machinery for LRO and lysosome exocytosis is likely dependent on the cell- type in question. In cells of the hemopoietic lineage, syntaxin 11 is required for the formation of the SNARE complex with vesicle associated membrane protein 8 (VAMP8) and SNAP-23 [514]. Mutations in syntaxin 11 leads to the development of familial hemophagocytic
lymphohistiocytosis (FHL) 4 [515]. Platelets and CTLs from FHL 4 patients have a deficit in LRO degranulation [514,516].
FHL can also be caused by mutations in Munc13-4 and Munc 18, which cause FHL 3 and FHL 5, respectively [517]. Munc 13-4 complexes with Rab27 and is critical for docking secretory lysosomes to the cell surface after stimulation. Mutation of critical residues that participate in Rab27 binding abrogates vesicle tethering to the cell surface and decreases lysosome secretion [517]. Munc 18-2 likely catalyzes membrane fusion between lysosomes and the cell surface. In synaptic vesicles, Munc 18 proteins are absolutely required for the fusion of synaptic vesicles with the presynaptic membrane [513]. In agreement, natural killer cells properly polarize
cytotoxic granules to the immunological synapse, but do not degranulate and result in decreased cell killing [518,519].
1.6
Rationale and Hypothesis
According the amyloid cascade hypothesis, the pathological production and accumulation of Aβ is thought to be the initiating factor in AD pathology [35]. Work from our lab and others have implicated the endosomal/lysosomal system in Aβ production [40,42,44,419]. Recent work has revealed that endosomes and lysosomes accumulate intraluminal Aβ [439,463,464,467]. These intracellular Aβ aggregates are resistant to degeneration and can seed the formation of Aβ fibrils in vitro [52,464].
In AD, the production and accumulation of Aβ appears to be a critical part of the disease process. Despite the importance of APP trafficking to lysosomes in production of Aβ, the process is not fully understood. The intracellular trafficking of nascent APP to the endosomal/lysosomal system is relatively understudied, due to the difficulty in tagging nascent proteins for
microscopy. APP also contains three tyrosine motifs, which have been shown to be involved in endocytosis and basolateral sorting [44,281]. However, the role of these motifs, if any, in the sorting of nascent APP from the Golgi apparatus is unknown. While it is thought that Aβ is produced intracellularly in lysosomes, the mechanism, which allows these intracellular stores to
access the extracellular space has yet to be elucidated. It is hypothesized that APP can traffic
intracellularly to lysosomes to produce Aβ, which can be secreted via lysosome exocytosis.