Nature Reviews Neuroscience | AOP, published online 11 February 2015; doi:10.1038/nrn3896

PROGRESS Retromer in Alzheimer disease, Parkinson disease and other neurological disorders Scott A. Small and Gregory A. Petsko

Abstract | Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of Alzheimer disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system. Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retro­mer’ was first introduced in a yeast study in 1998 (REF. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retro­ grade direction, from the yeast endosome back to the Golgi. By 2004, a handful of studies had identi­ fied the molecular 4 and the functional5,6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders. New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking 8. Synaptic function heav­ ily depends on endosomal trafficking, as

it contributes to the presynaptic release of neuro­transmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plastic­ ity 9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synap­ tic biology10–13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dys­function to several neurological diseases — notably, AD and Parkinson disease (PD). Function and organization The endosome is considered a hub for intra­ cellular transport. From the endosome, trans­ membrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (FIG. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as

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we describe, it is particularly important for the normal delivery of hydrolases and pro­ teases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14,15 (FIG. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other recep­ tors to the plasma membrane during synaptic remodelling and plasticity 10–13. As well as extending the endosomal trans­ port routes, recent studies have considerably expanded the number of molecular constitu­ ents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational com­ plexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each mod­ ule can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role. Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargorecognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5,6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19,20. Some studies suggest that VPS26a and VPS26b are functionally redundant 21, whereas others suggest that they might form distinct cargo-recognition cores20,22. The second central module of the ret­ romer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (FIG. 1b). The proteins in this module, which directly binds the cargo-rec­ ognition core, are members of the subgroup of the sorting nexin (SNX) family that are ADVANCE ONLINE PUBLICATION | 1

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PROGRESS a

b Recycling pathway

Cytosol

Tubule Actin

Endosome Retromer

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Retrograde pathway TGN

VPS35

Degradation pathway

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Cargorecognition core VPS26

Endosomal membrane

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Figure 1 | Retromer’s endosomal transport function and molecular organization.  a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal memNature Reviews | Neuroscience branes. The first is the retrograde pathway in PtdIns3P which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3‑phosphate.

characterized by the inclusion of a carboxyterminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (REFS 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combina­ tions, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (REFS 26,27). The EPS15‑homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28. A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabi­ lize the core once it is there (FIG. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (REF. 29), the RASrelated protein RAB7A30–32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16

(TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3‑phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15,34. SNX27 functions as an adap­ tor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection. Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21,

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strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28,35,36 (FIG. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to cre­ ate patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (REF. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway. The cargos that are transported by ret­ romer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co‑transported with the receptors. The receptors that are trans­ ported by retromer that have so far been iden­ tified to be the most relevant to neurological diseases are the family of VPS10 domaincontaining receptors (including sortilinrelated receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose‑6‑phosphate receptor (CIM6PR)6,5; glutamate receptors10; and phagocytic recep­ tors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β‑amyloid precursor protein (APP)7,38–41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane. Retromer dysfunction Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endo­ some for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid‑β, which is implicated in AD43 (FIG. 2a)). Second, by reduc­ ing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (FIG. 2b)). The third consequence (FIG. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5,6 or sortilin44, after these recep­ tors transport proteases from the TGN to www.nature.com/reviews/neuro

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APP Endocytosis pathway Endosome APP receptor Retromer

Amyloid-β βCTF

TGN

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APP processing

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CIM6PR

Reduced protease delivery

Tau aggregates Cathepsin D

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Figure 2 | The pathophysiology of retromer dysfunction.  Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β‑amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β‑carboxy-terminal fragment (βCTF) and amyloid‑β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose‑6‑phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal– lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

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the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway 5,6, allow­ ing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders. Although requiring further validation, recent studies suggest that retromer dys­ function might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the trans­ membrane protein autophagy-related pro­ tein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neu­ rons clear neurotoxic aggregates that accu­ mulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell‑to‑cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases. Alzheimer disease Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging stud­ ies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independ­ ent of ageing (as reviewed in REF. 51). At the same time, neuro­imaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individu­ als, intentionally covering a broad range of ages. A statistical analysis was applied to the ADVANCE ONLINE PUBLICATION | 3

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PROGRESS determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are dif­ ferentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (REF. 7). Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanisti­ cally linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expres­ sion levels correlated with VPS35 expression. The top ‘hit’ was the transcript encod­ ing the transmembrane protein SORL1 (REF. 43). As SORL1 belongs to the family of VPS10‑containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10‑containing proteins (sortil­ lin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co‑trafficked by retromer. This led to a model in which ret­ romer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP. Subsequent studies were required to fur­ ther establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the compo­ nents of the retromer cargo-recognition core and the family of VPS10‑containing recep­ tors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide associa­ tion study 54. Other genetic studies identified AD‑associated variants in genes encoding proteins that are linked to nearly all mod­ ules of the retromer assembly 55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encod­ ing proteins of the retromer membranerecruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033).

In addition, nearly all of the genes encod­ ing the family of VPS10‑containing ret­ romer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (REFS 7,37), all modules in the retromer assembly are implicated in AD. Studies in mice39,58,59, flies39 and cells in culture34,40,41,60,61 have investigated how ret­ romer dysfunction leads to the pathogenic processing of APP. Although rare discrep­ ancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dys­ function causes increased pathogenic pro­ cessing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10‑containing proteins function as APP receptors that mediate APP trafficking out of endosomes. Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54,63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor trigger­ ing receptor expressed on myeloid cells 2 (TREM2) (FIG. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduc­ tion in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors func­ tion in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease. The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biol­ ogy suggests several plausible mechanisms

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that are worth considering. Tau is a cyto­ solic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66,67. In fact, recent studies sug­ gest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau pro­ cessing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellu­ lar tau, this would accelerate tau endocytosis in neurons and its pathogenic processing. A second possibility comes from the estab­ lished role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (FIG. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is medi­ ated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endo­ some and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional pheno­ copy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunc­ tion and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions. Parkinson disease The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomaldominant mutations in VPS35 that cause late-onset PD69,70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear. Among a group of recent studies, all46,48,71–76 but one77 strongly suggest that these muta­ tions cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly www.nature.com/reviews/neuro

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PROGRESS but reduce the ability of VPS35 to associate with the WASH complex 46,75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71,73,75,76 from the endo­ some back to the TGN (FIG. 2c). In this sce­ nario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnor­ mal α‑synuclein processing and to the for­ mation of α‑synuclein aggregates, which are thought to have a key pathogenic role in PD. A separate study suggested that the muta­ tion might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophago­ somes46. Autophagosomes have also been implicated as an intracellular site in which α‑synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α‑synuclein aggregates48. Several studies in flies71,74 and in rat neu­ ronal cultures71 provide strong evidence that increasing retromer function by overexpress­ ing VPS35 rescues the neurotoxic effects of the most common PD‑causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neuro­ toxic effect of α‑synuclein aggregates in a mouse model48. These findings have imme­ diate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by the VPS35 muta­ tions or by knocking down VPS35 (REF. 71). Together, this and other studies78 suggest that LRRK2 might have a role in retromerdependent transport, but future studies are required to clarify this role. Other neurological disorders Besides AD and PD, in which a convergence of findings has established a strong patho­ genic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three dis­ orders for which the evidence of the involve­ ment of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syn­ drome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intel­ lectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the ret­ romer cargo-selection protein SNX27 might be a primary driver for some of these impair­ ments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer func­ tion, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications. Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by pro­ gressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex. The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84. Retromer as a therapeutic target As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this obser­ vation and after a decade-long search86, we

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identified a novel class of ‘retromer pharma­ cological chaperones’ that can bind and sta­ bilize retromer’s cargo-recognition core and increase retromer levels in neurons61. Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numer­ ous other pharmacological approaches for enhancing retromer function, this success provides the proof‑of‑principle that retromer is a tractable therapeutic target. As retromer functions in all cells, a gen­ eral concern is whether enhancing its func­ tion will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative con­ sequences in yeast, neuronal cultures, flies or mice40,48,61,71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic. If retromer drugs are safe and can effec­ tively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predeter­ mined evidence of retromer dysfunction. The most immediate example is that of individu­ als with PD that is caused by LRRK2 muta­ tions. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71,74 can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2‑linked PD might be an appropriate indication for clinical trials. Alternatively, the pathophysiology of a dis­ ease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of ret­ romer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial func­ tion, even if there are no pre-existing defects in retromer. More generally, histological studies com­ paring the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87,88. Importantly, enlarged endosomes are uni­ formly observed in a broad range of patients ADVANCE ONLINE PUBLICATION | 5

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PROGRESS with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathol­ ogy 87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89,90 — or too little cargo flowing out, as observed in retromer dysfunction40,61 and related transport defects57. By any mecha­ nism, retromer-enhancing drugs might cor­ rect this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology. Conclusions The fact that retromer defects, includ­ ing those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on ret­ romer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heav­ ily depends on endosomal transport and trafficking. Although plausible, future stud­ ies are required to confirm and to test the details of this hypothesis. However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the estab­ lished pathophysiological consequences of retromer dysfunction providing a mechanis­ tic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal popula­ tions? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer. The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be acrossthe-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to

be more circumscribed, involving selec­ tive disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87,88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91 suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria. Interestingly, studies suggest that the entorhinal cortex — a region that is dif­ ferentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensi­ tive to defects in endosomal transport in gen­ eral and retromer dysfunction in particular, and for why this region is the early site of dis­ ease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be par­ ticularly sensitive to the more circumscribed defect in retromer. Perhaps the most important observation for clinical neuroscience is the now wellestablished fact that increasing levels of ret­ romer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery 93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise. Scott A. Small is at the Taub Institute for Research on Alzheimer’s Disease and the Ageing Brain, Departments of Neurology, Radiology, and Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA. Gregory A. Petsko is at the Helen and Robert Appel Alzheimer’s Disease Research Institute, Department of Neurology and Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York 10065, USA. e‑mails: [email protected]; [email protected] doi:10.1038/nrn3896 Published online 11 February 2015

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Acknowledgements

The authors thank the US National Institute on Ageing, US National Institutes of Health grants AG025161 and AG08702, The Alzheimer’s Association, The McKnight Endowment for Neuroscience, the Ellison Medical Foundation and The Fidelity Biosciences Research Initiative for funding, and give special thanks to S. Weninger for advice and encouragement. The authors also thank G. Di Paolo for discussions regarding the manuscript.

Competing interests statement

The authors declare no competing interests.

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