More than Cholesterol Transporters: Lipoprotein Receptors in CNS Function and Neurodegeneration

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Review More than Cholesterol Transporters: Lipoprotein Receptors in CNS Function and Neurodegeneration Courtney Lane-Donovan,1 Gary T. Philips,1,2 and Joachim Herz1,3,* 1Department

of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA for Neural Science, New York University, New York, NY 10003, USA 3Departments of Neuroscience, Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2014.08.005 2Center

Members of the low-density lipoprotein (LDL) receptor gene family have a diverse set of biological functions that transcend lipid metabolism. Lipoprotein receptors have broad effects in both the developing and adult brain and participate in synapse development, cargo trafficking, and signal transduction. In addition, several family members play key roles in Alzheimer’s disease (AD) pathogenesis and neurodegeneration. This Review summarizes our current understanding of the role lipoprotein receptors play in CNS function and AD pathology, with a special emphasis on amyloid-independent roles in endocytosis and synaptic dysfunction. Introduction The low-density lipoprotein receptor (LDLR) family is a highly conserved receptor family with diverse functions in cellular physiology. The core members of the LDL receptor family include the LDL receptor (Yamamoto et al., 1984), LDLR-related protein 1 (LRP1) (Herz et al., 1988), the very-low-density lipoprotein receptor (VLDLR) (Takahashi et al., 1992), megalin (LRP2) (Saito et al., 1994), Apoer2 (LRP8) (Kim et al., 1996; Novak et al., 1996), LRP4 (Nakayama et al., 1998), and LRP1b (Liu et al., 2000). Orthologs of LDLR family members have been found in species throughout the animal kingdom, highlighting the fundamental role these receptors play in basic cellular processes. The members of the LDLR family share a conserved structure. At the C terminus, a short cytoplasmic tail contains one to three NPxY motifs, which facilitate signal transduction and endocytic trafficking through interactions with cytosolic adaptor proteins (Gotthardt et al., 2000). The extracellular domain has a varying number of complement-type repeats and epidermal growth factor precursor homology domains, which are required for ligand binding and pH-dependent ligand release, respectively (Davis et al., 1987; Esser et al., 1988). Additionally, the extracellular domain can interact with a variety of coreceptors. The lipoprotein receptors bind a wide variety of ligands with differing specificity, helping to explain the diversity of their actions. These ligands include, but are not limited to, developmental proteins (Sonic hedgehog [Christ et al., 2012], Wnt [Tamai et al., 2000], and Reelin [D’Arcangelo et al., 1999]), apolipoproteins (ApoE and ApoB), proteases and protease inhibitors (a2macroglobin [Strickland et al., 1990]), carriers of plasma vitamins (Moestrup and Verroust, 2001), chaperones (RAP [Kounnas et al., 1992]), and inflammatory mediators (TGFb [Muratoglu et al., 2011]). Through interactions with these ligands, the lipoprotein receptors have a diverse set of functions in the developing and adult nervous system, from lipoprotein trafficking, to synaptic plas-

ticity, to cell migration and development. Additionally, lipoprotein receptors affect neurodegenerative processes and are the key receptors for the main Alzheimer’s disease (AD) risk factor, ApoE4. This Review will touch on each of these aspects of lipoprotein receptor signaling in the nervous system. The Lipoprotein Receptors and Cholesterol Metabolism The lipoprotein receptors were initially identified for their role in peripheral cholesterol transport. Cholesterol is required for several processes in the body. It is a key component of the plasma membrane and a precursor for the production of steroid hormones. In the periphery, lipoprotein receptors bind triglyceride- and cholesterol-carrying lipoprotein particles such as chylomicrons, VLDL, and LDL for endocytosis via clathrin-coated pits into cells for further metabolism (Go and Mani, 2012). Similar to their role in the periphery, lipoprotein receptors mediate cholesterol transport in the CNS. Most plasma lipoproteins cannot cross the blood-brain barrier, and thus cholesterol-carrying particles are generally produced within the CNS (Bjo¨rkhem et al., 1998). In the CNS, glia-derived cholesterol is essential for the formation and maintenance of mature synapses (Mauch et al., 2001). Astrocytes release ApoE-containing ‘‘HDL-like’’ particles to transport cholesterol and phospholipids between glia and neurons (Boyles et al., 1985; Pitas et al., 1987). ApoE binds to a variety of cell surface receptors in the LDLR family for particle uptake, including LDLR, LRP1, VLDLR, and Apoer2 (Beffert et al., 2004). Lrp1 appears to have the highest transport capacity for ApoE, due to its rapid endoyctic recycling rates (Li et al., 2001). Moreover, selective deletion of Lrp1 in forebrain neurons leads to a global defect in brain lipid metabolism and neurodegeneration in mice (Liu et al., 2010). Other lipoprotein receptor family members may also participate in CNS cholesterol homeostasis. The importance of cholesterol homeostasis for brain function is highlighted by Niemann-Pick type C (NPC) disease. In NPC, autosomal recessive mutations in NPC1 or NPC2 result in Neuron 83, August 20, 2014 ª2014 Elsevier Inc. 771

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Review Figure 1. ApoE, Reelin, and Synaptic Plasticity (A) Reelin binds to its receptor, Apoer2, activating a signaling cascade through Dab1 and SFKs to phosphorylate NMDA receptors, resulting in greater Ca2+ influx upon glutamate signaling (Benhayon et al., 2003; Chen et al., 2005). Dab1 also acts to counteract Ab and PP2B’s effects on Akt, GSK3b, and tau phosphorylation (Beffert et al., 2002; Durakoglugil et al., 2009). (B) Reelin signaling results in the endocytosis of Apoer2, AMPA receptors, and NMDA receptors. ApoE isoforms differentially affect the recycling of these endosomes (Chen et al., 2010). ApoE3-containing endosomes readily recycle back to the surface, while those containing ApoE4 remain trapped (trapped vesicles are displayed at reduced size). (C) Apoer2 and EphB2 bind the NR2 and NR1 subunits of NMDAR, respectively, regulating receptor function and endocytosis (Beffert et al., 2005; Dalva et al., 2000). Reelin binds to EphB2 and Apoer2 through independent, distinct sites (Bouche´ et al., 2013). Akt, protein kinase B; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; ApoE, apolipoprotein E; Apoer2, apolipoprotein E receptor 2; Dab1, disabled-1; glu, glutamate; GSK3b, glycogen synthase kinase 3 beta; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate; PI3K, phosphoinositide 3-kinase; PP2B, protein phosphatase 2B, calcineurin; SFKs, Src-family kinases.

impaired cholesterol trafficking, which leads to a buildup of cholesterol and lipids in the lysosome and a deficiency of these molecules for use both in the membrane and as a precursor for steroid synthesis (Carstea et al., 1997; Naureckiene et al., 2000). NPC is characterized primarily by progressive neurodegeneration, which indicates the brain’s high requirement for cholesterol relative to other organs. Lipoprotein Receptors and the Developing Brain Several lipoprotein receptors play a vital role in the developing embryo. Megalin, or Lrp2, is a 600 kDa lipoprotein receptor expressed predominantly in the developing embryo and the mammalian kidney (Saito et al., 1994). Lrp2 is required for normal organogenesis, and Lrp2 knockout mice have holoprosencephaly with fused hemispheres and no olfactory bulbs (Willnow et al., 1996). This phenotype results from increased BMP4 expression and a reduction of sonic hedgehog (Shh) expression, which leads to a loss of interneurons and oligodendroglial cell populations (Spoelgen et al., 2005). Individuals with autosomal recessive loss-of-function mutations in LRP2 develop DonnaiBarrow syndrome, which is characterized by facial dysmorphology, ocular and hearing anomalies, and agenesis of the corpus callosum (Kantarci et al., 2007). The lipoprotein receptors Apoer2 and Vldlr also affect development as mediators of Reelin signaling. Reelin is a large secreted extracellular matrix protein, released early in development by Cajal-Retzius cells and later in life by a subset of GABAergic interneurons (Curran and D’Arcangelo, 1998). Reelin signaling is required for proper neuronal migration and maturation, and Reelin knockout (reeler) mice have inverted cortical layering, cerebellar hypoplasia, and immature synapses (Falconer, 1951; Niu et al., 2008). Reeler mice also have impaired learning and motor coordination as well as severe neurodevelopmental deficits that lead to strain background-dependent lethality by about 5 weeks of age (D’Arcangelo et al., 1995). Since Reelin uses both Apoer2 and Vldlr to mediate its signaling, Apoer2 and Vldlr double knockout mice also have a reeler phenotype (Trommsdorff et al., 1999). Patients with mutations in RELN or VLDLR have been identified 772 Neuron 83, August 20, 2014 ª2014 Elsevier Inc.

that recapitulate certain aspects of the reeler phenotype (Hong et al., 2000; Ozcelik et al., 2008), which underscores the importance of these receptors in the development of the CNS. Lipoprotein Receptors, Reelin, and Synaptic Plasticity Several of the lipoprotein receptors are expressed in the adult CNS. Lrp1 is expressed in astrocytes, microglia, and neurons in the postsynaptic density (May et al., 2004). VLDLR is expressed by many cell types, and in the brain it is found in glia, neuroblasts, matrix cells, and pyramidal neurons on cell membranes outside of lipid rafts (Christie et al., 1996; Duit et al., 2010). Similarly, LDLR is expressed in both astrocytes and neurons (Fan et al., 2001). Conversely, Apoer2 is largely restricted to the testes and brain, where it is expressed in neurons throughout the CNS and traffics into the postsynaptic density (Beffert et al., 2005; Clatworthy et al., 1999). Due to their localization and expression in neurons, lipoprotein receptors are well-placed to affect synaptic function and synaptic plasticity. The lipoprotein receptors Apoer2 and Vldlr play a role in synaptic plasticity through their interaction with the glycoprotein Reelin. At the adult synapse, Reelin has a key effect of enhancing longterm potentiation (LTP). Reelin binding clusters Apoer2 and Vldlr, leading to receptor dimerization and tyrosine phosphorylation of the cytosolic protein Dab1 (Benhayon et al., 2003; D’Arcangelo et al., 1999; Herz and Chen, 2006; Hiesberger et al., 1999; Howell et al., 1997; Strasser et al., 2004). Dab1 then activates Src tyrosine kinases (SFKs), including Fyn, which phosphorylate NMDA receptors, enhancing their Ca2+ influx (Chen et al., 2005). This increased Ca2+ influx allows for a stronger NMDA current at similar levels of glutamate release, resulting in greater AMPA receptor insertion into the membrane and enhanced LTP (Weeber et al., 2002; Figure 1A). Consistent with its ability to enhance LTP, acute intraventricular Reelin injections improve memory performance of wild-type mice and can rescue learning and memory deficits in heterozygous reeler mice (Rogers et al., 2011, 2013). The robust effect of Reelin signaling has led to a search for other Reelin receptors. In addition to Apoer2 and Vldlr, Reelin also binds EphB2, a member of the EphB receptor family

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Review (Bouche´ et al., 2013). EphB receptors are required for proper CNS development and synaptic function (Klein, 2009; Xu and Henkemeyer, 2012). EphB2 activation by its ligand Ephrin-B1 results in the clustering of EphB2 and NMDA receptors and promotes excitatory synaptic development (Dalva et al., 2000). In mature synapses, EphB2 promotes synaptic targeting of NR2B subunits, which allows greater calcium influx upon NMDAR activation and may shift the synaptic plasticity balance toward LTP (Nolt et al., 2011). In vitro, Reelin can at least partially activate EphB2 signaling; however, the effects of Reelin-mediated EphB2 signaling on NMDA receptor targeting and adult synaptic function have yet to be determined (Bouche´ et al., 2013). Intriguingly, Apoer2 binds NR2 to promote its phosphorylation, while EphB2 binds NR1 (Beffert et al., 2005; Dalva et al., 2000; Hoe et al., 2006). Reelin binds to each of these receptors at a distinct and separate site, suggesting that Apoer2, NMDAR, and EphB2 may form a macromolecular complex at the synapse (Bouche´ et al., 2013) (Figure 1C). In addition to EphB receptors, it has been reported that LDLR may interact with Reelin (D’Arcangelo et al., 1999), albeit much more weakly than Apoer2 and Vldlr. Functionally, these are the main Reelin receptors, since Apoer2/Vldlr double knockout mice are phenotypically identical to reeler mice (Trommsdorff et al., 1999). Most studies of the synaptic role of Reelin have focused on its postsynaptic effects; however, new research suggests that Reelin also affects the release of VAMP7-containing synaptic vesicles (Bal et al., 2013). Previously, Apoer2 and Vldlr had been thought to be predominantly postsynaptic proteins. Intriguingly, reeler mice have more vesicles at the presynaptic bouton and a reduction in SNAP25, an element of the vesicle fusion machinery, suggesting impairment in vesicle fusion and release. However, this effect is not dependent on either Apoer2 or Vldlr but rather the integrin pathway (Hellwig et al., 2011). The precise details by which Reelin signals at the presynapse and the role of Reelin receptor signaling remain to be clarified. The Role of Lipoprotein Receptors in the Peripheral Nervous System The neural function of lipoprotein receptors is not limited to the brain; in fact, they play a large role in the development of the neuromuscular junction (NMJ). The vertebrate NMJ is a specialized cholinergic synapse in which presynaptic motor neurons connect with postsynaptic muscle fibers at the motor endplate. The lipoprotein receptor Lrp4 plays several roles in NMJ formation. First, Lrp4 forms a complex with MuSK to prepattern the muscle (Kim and Burden, 2008). Activation of MuSK results in the recruitment of several downstream pathways that result in nicotinic acetylcholine receptor (AChR) clustering. When the motor neuron arrives, it releases agrin, which stimulates MuSK to promote maturation of the synapse. Lrp4 is required for the relay of the agrin signaling to MuSK and in the absence of Lrp4, agrin’s ability to induce MuSK phosphorylation is minimal (Kim et al., 2008; Zhang et al., 2008). Finally, independent of agrin or MuSK, Lrp4 acts as a retrograde messenger to promote presynaptic vesicle clustering (Yumoto et al., 2012). Surprisingly, Lrp4 only seems to be required for NMJ development in some mammalian species. While Lrp4 knockout mice exhibit neonatal lethality due to failure to form the NMJ (Weatherbee et al., 2006),

homozygous mutations in LRP4 that would be predicted to abolish its functionality cause Cenani-Lenz syndrome in humans and mulefoot disease in cattle (Johnson et al., 2006; Karner et al., 2010; Li et al., 2010), with apparently functional NMJs. These findings could point to a different mechanism in the organization of the NMJ in cattle and humans or a redundancy that may compensate for functional Lrp4 loss. This redundancy could potentially be mediated by Lrp10, an LDLR family member that is virtually identical to Lrp4; however, it remains to be determined whether LRP10 indeed arose from a recent gene duplication. The amyloid precursor protein, APP, which is discussed in depth later in the next section for its role in Alzheimer’s disease, also takes part in NMJ development. APP, or its paralogue APLP2, is required both pre- and postsynaptically for NMJ development (Akaaboune et al., 2000; Wang et al., 2005). One hypothesis is that APP acts transsynaptically to promote crosstalk between the pre- and postsynaptic compartments. While this has not been conclusively shown, it is known that APP can bind both to itself and to APLP2 in trans to promote cell adhesion in vitro (Soba et al., 2005). At the presynaptic side, APP interactions promote choline transporter (CHT) activity and expression, and they promote L-type calcium channel function, both of which are required for proper NMJ function (Wang et al., 2007; Yang et al., 2007). Finally, APP acts postsynaptically on the motor endplate to promote Lrp4 and MuSK interaction (Choi et al., 2013). These findings at peripheral synapses may also apply at central synapses and point the way for future studies on the role of lipoprotein receptors in central synapse development. Lipoprotein Receptors and Neurodegenerative Disease Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by progressive memory loss and a pathological accumulation of amyloid plaques and neurofibrillary tangles. Considerable research into the disease has focused on the genetic, cognitive, and pathological changes found in individuals with early-onset familial disease, who represent approximately 1% of the patient population. These individuals have mutations in either the amyloid precursor protein (APP) or its cleavage proteins PS1 and PS2, resulting in the early accumulation of plaqueforming amyloid b (Ab) (Weggen and Beher, 2012). However, the majority of AD patients have late-onset disease (LOAD), with no mutations in the Ab-generating machinery. The major genetic risk factor for LOAD is the ε4 isoform of apolipoprotein E—a cholesterol transport protein. In humans, ApoE exists in three isoforms that differ at two residues: ε2 (ApoE2 with cys112 and cys158), ε3 (ApoE3 with cys112 and arg158), and ε4 (ApoE4 with arg112 and arg158). These variations confer significant differences in disease risk. ApoE3 is the most common isoform and thus is considered the gold standard from the population genetic risk perspective. Relative to ApoE3, ApoE4 is present in approximately 15%– 20% of the population and is associated with greatly increased risk and earlier onset of AD (Corder et al., 1993; Schmechel et al., 1993). Meanwhile, the ApoE2 isoform is protective against AD relative to ApoE3 and ApoE4 (Corder et al., 1994). The structural differences between these isoforms contribute to differences in protein conformation, receptor binding, and endocytic Neuron 83, August 20, 2014 ª2014 Elsevier Inc. 773

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Review trafficking (reviewed in Kanekiyo et al., 2014). Interestingly, genetic association studies of polymorphisms in lipoprotein receptors with AD risk have only revealed potential minor effects of LRP1 (Beffert et al., 1999; Jin et al., 2013). This may be due to the fact that several of the lipoprotein receptors have key developmental roles, which could preclude major effects on neurodegeneration. By contrast, a significant association of SORL1 with AD has been reported (Rogaeva et al., 2007), which has been corroborated mechanistically on the molecular level (Schmidt et al., 2012). What links lipoprotein receptors to AD? The difficulty in finding the answer stems from the fact that late-onset AD is a multifactorial disease involving endocytic dysfunction, lipoprotein signaling, and synaptic dysregulation—essentially three different disciplines. The remainder of this Review focuses on the variety of roles played by lipoprotein receptors in neurodegenerative processes. APP Processing and the Endosome The pathological hallmark of Alzheimer’s disease is progressive accumulation of amyloid plaques. These plaques are formed of aggregated amyloid b (Ab), the end product of sequential cleavage of amyloid precursor protein (APP) (reviewed in Zhang et al., 2012). Briefly, APP is cleaved first by b-secretase (BACE-1) to sAPPb and b C-terminal fragment (bCTF) (Cai et al., 2001). bCTF is then cleaved by g-secretase: a complex of multiple proteins that includes presenilin 1 or 2 (PS1 or PS2), APH, nicastrin, and PEN2 (St George-Hyslop and Fraser, 2012), resulting in the final cleavage product, Ab. The APP metabolites resulting from this sequential cleavage play their own roles in neuronal function, reviewed in depth in (Chow et al., 2010). sAPPb, generated by b-cleavage, appears to be involved in synaptic pruning and apoptosis (Nikolaev et al., 2009). In contrast to amyloidogenic b-cleavage, APP can be cleaved by a-secretase within the Ab segment to generate the nonamyloidogenic sAPPa (Vingtdeux and Marambaud, 2012). sAPPa is generally considered to be neuroprotective, enhancing LTP and spatial learning (Taylor et al., 2008). The other cleavage products created by a- and b-cleavage, CTF83 and CTF99, respectively, do not have well-defined roles. However, g-secretase cleavage of the CTFs results in the release of the APP Intracellular Domain (AICD), which binds Fe65 and TIP60 for potential transcriptional activation of downstream targets (Cao and Su¨dhof, 2001), ultimately promoting cell death and impairing neurogenesis (Ghosal et al., 2010; Wang et al., 2014). The final and most-studied products of APP cleavage are the Ab species, predominantly Ab40 and some Ab42. Of these two species, Ab42 is considered to be more amyloidogenic and thus more toxic (Aoki et al., 2008; Kim et al., 2007; McGowan et al., 2005). Mutations causing early-onset Alzheimer’s disease directly affect this process—mutations in APP facilitate cleavage by BACE1 to amyloidogenic species, while mutations in PSEN1 and PSEN2 enhance their g-cleavage ability and tend to produce a higher Ab42:Ab40 ratio (Weggen and Beher, 2012). By contrast, in LOAD, there is no inherent dysfunction of the key enzymes of Ab generation, yet Ab still accumulates. Whether APP is a or b-cleaved is determined by the localization of APP processing proteins. a-secretase is active at the mem774 Neuron 83, August 20, 2014 ª2014 Elsevier Inc.

brane surface, whereas BACE1 is most active at the lower pH found in endosomes (Cole and Vassar, 2007). As a result, conditions that result in increased trafficking of APP to the endosome are pro-amyloidogenic, whereas those that maintain APP at the surface are anti-amyloidogenic. Moreover, in healthy brains, APP and BACE1 are largely localized to different compartments, while AD brains show increased colocalization of the two proteins within acidic microdomains (Das et al., 2013). Additionally, overexpression of ApoE4 promotes colocalization of APP and BACE1 in vitro (Rhinn et al., 2013). However, localization to the membrane surface is not enough to prevent APP b-cleavage. Lipid rafts in the membrane also have increased BACE1 activity and decreased a-secretase activity (Ehehalt et al., 2003). It is likely that b-cleavage in lipid rafts contributes to Ab generation; however, the bulk of Ab generation, approximately 70%, is dependent on endocytic recycling (Cirrito et al., 2008). Histological evidence suggests that dysfunction of endocytic recycling is one of the earliest pathological changes in AD. In postmortem studies of AD patients, early endosomes were enlarged up to 32-fold the volume of normal endosomes (Cataldo et al., 1997). In theory, the enlarged endosomes give APP more time to interact with b- and g-secretases and thus generate more Ab. Remarkably, endosomal enlargement begins prior to the appearance of clinical disease in APOE ε4 carriers, and this finding was reproduced in induced pluripotent stem cells derived from AD patient fibroblasts (Cataldo et al., 2000; Israel et al., 2012). Partly because of these findings, recent AD research has focused on changes in endosome recycling associated with the members of the LDL receptor family. Several members of the lipoprotein receptor family affect APP endocytosis. For example, LRP1 has an endocytosis rate that is much faster than APP. As a result, when the LRP1 extracellular domain interacts with APP, APP endocytosis is accelerated, leading to increased processing to Ab (Cam et al., 2005). Overexpression of a functional LRP1 minireceptor led to increased soluble Ab levels in the PDAPP mouse model (Zerbinatti et al., 2004, 2006). However, LRP1 also plays a role in promoting neuronal Ab clearance and neuronally restricted disruption of Lrp1 in APP/PS1 model mice accelerated Ab accumulation (Kanekiyo et al., 2013). Additionally, the scaffolding protein RanBP9 increases APP localization to lipid rafts and enhances the association of LRP1, APP, and BACE1, to increase Ab generation (Lakshmana et al., 2009). RanBP9 is increased in AD brains and in APP mice, suggesting that it may have a role in disease pathogenesis (Lakshmana et al., 2010; Woo et al., 2012). A newly identified member of the LDLR family, low-density-lipoprotein receptor class A domain containing 3 (LRAD3), also binds to APP and promotes amyloidogenic processing (Ranganathan et al., 2011). In contrast to LRP1 and LRAD3, the related receptor LRP1B has a slower rate of endocytosis and retains APP at the cell surface, which leads to decreased Ab generation (Cam et al., 2004). Similarly, LRP10, another novel LDLR family member, binds to APP and promotes its trafficking to the Golgi complex, which also prevents amyloidogenic processing (Brodeur et al., 2012). The role of the LDL receptor family member Apoer2 in the endocytic processing of APP is less clear. Apoer2 alone, in the absence of ligand, can cause APP localization to lipid rafts,

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Review Figure 2. APP Processing and Endocytosis In the proamyloidogenic pathway, APP is cleaved sequentially by BACE1 to sAPPb and C99 (Cai et al., 2001), and then sAPPb is cleaved by g-secretase to Ab and AICD (St George-Hyslop and Fraser, 2012). In the nonamyloidogenic pathway, a-secretase cleaves APP through the Ab region to generate C83 and sAPPa, which is cleaved to P3 and AICD by g-secretase (Vingtdeux and Marambaud, 2012). b-cleavage is favored in the endosome and in lipid rafts, while a-cleavage is favored at the cell surface outside of lipid rafts (Ehehalt et al., 2003). Several members of the low-density lipoprotein receptor family affect APP localization. LRP1 increases APP trafficking to the endosome (Cam et al., 2005), while LRP1B decreases trafficking (Cam et al., 2004). Apoer2 can direct APP to lipid rafts independently or to a-secretase in the presence of F-spondin (Hoe et al., 2005). SORLA can prevent a- and b-cleavage and promotes APP transport to the Golgi (Andersen et al., 2005; Schmidt et al., 2007). Ab, amyloid beta; AICD, APP intracellular domain; Apoer2, apolipoprotein E receptor-2; APP, amyloid precursor protein; Arc, activity-regulated cytoskeleton-associated protein; BACE1, beta-site APP cleaving enzyme 1; C83, APP C-terminal C83 fragment; C99, APP C-terminal C99 fragment; LRP1, low-density lipoprotein receptor-related protein 1; LRP1B, low-density lipoprotein receptor-related protein 1B; sAPPa, soluble APP a; sAPPb, soluble APP b; SORLA, sortilin-related receptor.

which leads to increased Ab generation in the absence of endocytosis (Fuentealba et al., 2007). In the presence of F-spondin, which interacts with Apoer2 and APP, Apoer2’s slower endocytosis rate inhibits APP endocytosis, maintaining APP at the surface and reducing Ab generation (Hoe et al., 2005). However, ApoE binding to Apoer2 causes endocytosis of APP, Apoer2, and BACE1 (He et al., 2007). Together, these data suggest that the effect of Apoer2 on APP endocytosis depends on the ligands present. Intriguingly, VLDLR and APP can increase each other’s surface expression through mutual interactions with Fe65 (Dumanis et al., 2012). In addition to the LDL receptor gene family of ApoE receptors, members of the structurally related Vps10p domain-containing receptor family are also involved in APP trafficking. Sorting protein-related receptor with A-type repeats (SorLA, alternatively SORL1 or LR11) is a neuronal Vps10p-containing receptor that interacts with APP and generally reduces its cleavage by either a- or b-secretases (Schmidt et al., 2007). Human studies have shown that SorLA is decreased in late-onset AD, and certain genetic variants of SORL1 are associated with AD, indicating a possible pathogenic role for SorLA (Dodson et al., 2006; Rogaeva et al., 2007; Scherzer et al., 2004). Recent data support a novel inhibitory mechanism for SorLA, where it binds to APP monomers and prevents them from forming dimers, which are the preferred ligands for both a- and b-secretases (Schmidt et al., 2012). Overexpression of SorLA causes the redistribution of APP to the Golgi complex, where all secretases are less active, leading to a decrease in Ab levels (Andersen et al., 2005). Recently, another member of the Vps10p family, sortilin, was identified as being involved with APP endocytosis in vitro. Sortilin promotes the endocytosis of APP and the generation of sAPPa (Gustafsen et al., 2013). However, an in vivo study in sortilin knockout mice did not reveal a change in sAPPa or sAPPb levels (Carlo et al., 2013) (Figure 2).

Alterations in endocytosis of APP clearly play a role in the pathology of AD, based on evidence from patient tissue and mouse models of disease. However, it is still not entirely clear what role ApoE isoforms play physiologically in APP endocytosis and subsequent Ab generation. In one study, the ApoE4 isoform caused increased endocytosis of APP, Apoer2, and BACE1 in neuroblastoma cells (He et al., 2007). Another study showed that ApoE4 delayed recycling of endosomes in hepatocytes (Heeren et al., 2004) and neuronal cells (Rellin et al., 2008). However, in vivo studies using microdialysis of the interstitial fluid (ISF) in ApoE transgenic mice showed that ApoE isoform has no effect on Ab synthesis, only its clearance (Castellano et al., 2011). Synaptic Activity Increases Ab Generation The physiological role of Ab has long been debated. A few studies have demonstrated that blocking Ab in young mice leads to reduced performance on memory tasks, suggesting that at low levels Ab plays a role in synaptic function (Abramov et al., 2009; Morley et al., 2010; Puzzo et al., 2008, 2011). Remarkably, synaptic activity increases levels of extracellular Ab and decreases levels of intraneuronal Ab (Cirrito et al., 2005). This is achieved by two separate mechanisms. First, the activitydependent generation of extracellular Ab is dependent on APP endocytosis and processing by BACE1 and g-secretase (Cirrito et al., 2008; Kamenetz et al., 2003). This is in part mediated by Arc, an intermediate early gene that is expressed shortly following synaptic activity (Soule´ et al., 2008). Arc increases the association of g-secretase with APP in endosomes (Wu et al., 2011). Conversely, synaptic activity decreases intraneuronal Ab42 through increased processing by neprilysin, the main Ab protease (Tampellini et al., 2009). Intriguingly, these results suggest a physiological role for Ab. In response to synaptic activity, Ab is increased extracellularly, which dampens LTP at Neuron 83, August 20, 2014 ª2014 Elsevier Inc. 775

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Review dendrites in the area through a number of candidate receptors including NMDA receptors, mGluR5, and PrPc (Laure´n et al., 2009; Li et al., 2011a; Renner et al., 2010). This could indicate that Ab is involved in homeostatic scaling, a process in which highly active synapses begin to dampen their responses, and less active ones become more sensitive (reviewed in Sheng et al., 2012). Reelin, Ab, and Tau at the Synapse Ab oligomers have been shown to inhibit LTP induction (Nomura et al., 2012; Shankar et al., 2008; Walsh et al., 2002). Ab activates caspase-3 through a mechanism that is currently unknown but probably involves mitochondrial dysfunction. Caspase-3 then activates PP2B (calcineurin), which directly dephosphorylates NMDAR at the Fyn phosphorylation site (D’Amelio et al., 2011; Kim et al., 2009; Snyder et al., 2005). Caspase-3 also cleaves Akt, rendering Akt inactive and unable to phosphorylate and inhibit GSK3b (Jo et al., 2011). When left uninhibited, GSK3b blocks LTP induction by an unknown mechanism that may involve tau (see below). Reelin has the opposite effect from Ab on GSK3b activity. Dab1 activates PI3K, which activates Akt, leading to decreased GSK3b activation (Beffert et al., 2002). Convincingly, Ab inhibition of LTP in hippocampal slices is prevented by coapplication of Reelin in the ACSF (Durakoglugil et al., 2009). Thus, Reelin signaling through ApoE receptors intersects with Ab at the level of tyrosine phosphorylation of NMDA receptors and GSK3b activation to affect LTP induction, learning, and memory (Figure 1A). As previously mentioned, the two main Reelin receptors, Apoer2 and Vldlr, also bind ApoE. Different ApoE isoforms differentially affect Reelin’s ability to signal through the ApoE receptors by interfering with receptor recycling to and from the synapse. Once Reelin activates its receptors, the receptors are endocytosed, along with Reelin and ApoE. While ApoE2 and ApoE3 do not significantly interfere with endosomal recycling, ApoE4 impairs it. This impaired recycling decreases the available surface pool of Apoer2 and thus Reelin signaling (Figure 1B). As a result, hippocampal slices from ApoE4 transgenic mice do not enhance LTP upon Reelin addition to the perfusate (Chen et al., 2010). Importantly, because Reelin is unable to efficiently signal in ApoE4 knockin mice, Reelin is unable to protect against the Ab-induced synaptic suppression. Consequently, ApoE4 promotes increased susceptibility to Ab damage at the synapse (Chen et al., 2010). The importance of Reelin in AD pathology is implicated by several studies. Reelin levels are reduced in the entorhinal cortex in postmortem AD brain and hAPP mouse models (Chin et al., 2007; Herring et al., 2012). Interestingly, another group found that Reelin levels were elevated in frontal cortex of AD patients, suggesting potential compensation in that region for elevated Ab levels (Botella-Lo´pez et al., 2006, 2010). Additionally, cognitively normal individuals with a high level of plaques and tangles possess certain SNPs in RELN that may make them more prone to elevating Reelin levels in response to high Ab load (Kramer et al., 2011; for review, see Krstic et al., 2013). Finally, Reelin is not the only ligand that binds to Apoer2 and Vldlr to activate Dab1 signaling. Clusterin, aka ApoJ, also binds to Apoer2 and Vldlr and can activate Reelin signaling (Andersen et al., 2003; 776 Neuron 83, August 20, 2014 ª2014 Elsevier Inc.

Leeb et al., 2014). Clusterin SNPs are associated with AD, and clusterin levels are elevated in AD (Harold et al., 2009; Lambert et al., 2009; May et al., 1990), further highlighting the role of ApoE receptors in AD pathogenesis. As described earlier, activation of the Reelin pathway culminates in GSK3b inhibition. In many disease states, including Alzheimer’s disease, GSK3b is unregulated, leading to its overactivation (Jope et al., 2007). One proposed mechanism for GSK3b’s effects on synaptic plasticity is tau hyperphosphorylation. Aggregated, hyperphosphorylated tau comprises the second pathological hallmark of AD, neurofibrillary tangles (NFTs). In a diseased brain, tau becomes phosphorylated at several sites by a number of kinases, leading to its detachment from the microtubule (Go¨tz et al., 2010). Hyperphosphorylated tau migrates from the axonal compartment to the somatodendritic compartment, where it destabilizes the synapse (Hoover et al., 2010; Li et al., 2011b). Tau aggregates to form NFTs, ultimately resulting in neuronal death. Remarkably, activated GSK3b is one of the kinases that phosphorylates tau, indicating a potential mechanism for its inhibition of LTP (Mulot et al., 1994). Indeed, hippocampal slices from tau knockout mice are resistant to Ab-induced LTP reduction (Shipton et al., 2011), as well as learning and memory deficits (Roberson et al., 2007). The effect of Reelin pathway dysfunction on tau phosphorylation appears to be strain specific. While some mouse strains have high levels of tau hyperphosphorylation in the absence of Dab1, other strains are relatively unaffected. A quantitative trait locus analysis comparing Dab1-deficient mice with high or low levels of tau phosphorylation revealed a strong hit on chromosome 16 at a locus that includes the genes for APP and SOD, as well as a suggestive hit at the locus that includes Psen1 (Brich et al., 2003). The genetic interplay between these key proteins highlights the integrative effects of the Ab, Reelin, and tau pathways in disease pathogenesis. The process by which dendritic tau causes synaptic dysfunction is largely unknown. One hypothesis is that tau acts as a carrier for Fyn kinase to the dendrites. At the synapse, Fyn kinase phosphorylates the NR2 subunit of NMDA receptors, which facilitates its interaction with PSD-95, and may lead to increased glutamate excitotoxicity. In the absence of tau, or with a truncated version of tau incapable of entering the dendrite (Dtau), Fyn remains localized to the soma. As a result, APP23 mice with no tau or Dtau have fewer memory deficits despite having similar levels of APP and Ab as those with full-length tau (Ittner et al., 2010). Fyn’s role in tau pathology is supported by the fact that overexpression of Fyn leads to greater impairment in spatial learning in APP mice (Chin et al., 2005). However, the tau-Fyn hypothesis, reviewed in depth in Ittner and Go¨tz (2011), is seemingly at odds with the physiological requirement for Fyn at the synapse and with the proposed mechanism of Reelin enhancement of LTP (Grant et al., 1992). As described above, Reelin activates Fyn kinase, but this enhances LTP, rather than causing excitotoxicity. The answer to this conundrum may lie in a different compartmentalization of these effectors: for example, the effect of Ab and tau on extrasynaptic versus synaptic NMDA receptors, and the role of NR2B versus NR2A subunits (Hardingham et al., 2002; Tackenberg et al., 2013; Talantova et al., 2013; see Parsons and Raymond, 2014 for a review).

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Figure 3. Removing Ab from the CNS Ab can be degraded by IDE in the ISF or taken up into microglia for degradation by neprilysin (Nep) (Jiang et al., 2008; Qiu et al., 1998). Ab can also be cleared across the BBB to the plasma, where it is degraded by plasma proteases. This process is affected by Ab binding to ApoE particles. While ApoE3 uses both Vldlr and LRP1 to cross the BBB, ApoE4 primarily uses Vldlr, which results in slower efflux (Deane et al., 2008). Lipidated ApoE is more effective at transporting Ab across the BBB than alipidated forms (Jiang et al., 2008). ABCA1 is a membrane protein that lipidates ApoE particles (Lawn et al., 1999) and whose expression is upregulated by LXR and RXR (Beaven and Tontonoz, 2006). Finally, when the tight junctions of the BBB are weakened, Ab can leak back into the ISF (Clifford et al., 2007). Ab, amyloid beta; ABCA1, ATP-binding cassette transporter; AD, Alzheimer’s disease; ApoE, apopolipoprotein E; APP, amyloid precursor protein BBB, blood-brain barrier; IDE, insulindegrading enzyme; ISF, interstitial fluid; LRP1, low-density lipoprotein receptor-related protein 1; LXR, liver X receptor; RXR, retinoid X receptor; Vldlr, very-low-density lipoprotein receptor.

Clearly, more work is required to better understand the interactions of Ab, tau, and Reelin at the synapse. ApoE and Ab Clearance Once Ab is generated by secretase cleavage, it is released to the ISF where it is cleared by a variety of mechanisms: efflux across the blood-brain barrier (BBB), uptake by cells for lysosomal degradation, and cleavage by Ab-specific proteases. Dysfunctional clearance of Ab plays a key role in amyloid accumulation, since late-onset AD patients do not have increased Ab production but rather insufficient Ab clearance (Mawuenyega et al., 2010). Further, Ab42 is generally cleared more slowly than Ab40, which leads to a preferential accumulation of the more toxic species (Bell et al., 2007). Ab is cleared from the ISF in an ApoE-isoform dependent manner: ApoE2 allows for the more rapid clearance of Ab in vivo, while ApoE4 slows clearance. Thus, the presence of ApoE4 promotes the buildup of Ab in the ISF (Castellano et al., 2011). The ApoE receptors LRP1, LDLR, and VLDLR all appear to play a key role in the clearance of Ab. As these Ab-clearing effects of ApoE were recently reviewed in depth (Kanekiyo et al., 2014), we will only touch on them briefly here.

One method of Ab clearance is efflux across the BBB to the periphery, where Ab is rapidly degraded in the plasma. ApoE receptors directly mediate this process, primarily LRP1, which uses its ligands, a2-macroglobulin, RAP, or ApoE, to promote Ab efflux (Bell et al., 2007; Hughes et al., 1998; Kanekiyo and Bu, 2009; Narita et al., 1997). ApoE2 and ApoE3 use both LRP1 and Vldlr to cross the BBB, leading to faster Ab efflux. ApoE4 shifts Ab transport from LRP1 to Vldlr, resulting in a slower rate of clearance and buildup of Ab in the ISF (Deane et al., 2008). The LDLR was also recently found to promote Ab efflux across the BBB (Castellano et al., 2012). While it is clear that lipoprotein receptors have a large effect on Ab efflux at the BBB, further work is required to elucidate their exact mechanisms of action. Normally, transport across the BBB is highly regulated by transporters such as the lipoprotein receptors; however, if the BBB is damaged, this regulation is lost. Perhaps unsurprisingly, the BBB is found to be defective in some, but not all, postmortem AD samples (Clifford et al., 2007). If the BBB has been damaged, Ab species may leak back into the CNS and accumulate in neurons, contributing to amyloid pathology (Clifford et al., 2007). In vitro experiments disagree on the role of ApoE and its isoforms on BBB maintenance (Hafezi-Moghadam et al., 2007; Nishitsuji et al., 2011). The importance of ApoE4 in BBB stability in vivo needs to be clarified, since only some AD patients have a weakened BBB, and the deterioration might be a result of disease pathology, rather than the cause. In addition to efflux across the BBB, Ab can be cleared centrally by endocytic trafficking to lysosomes or degradation by Ab proteases. ApoE3 promotes Ab uptake and degradation by lysosomes in vitro, while ApoE4 has a weaker effect (Li et al., 2012). In addition to lysosomal proteases, there are two Ab-specific proteases. Intracellularly, the degradation of Ab is mediated by neprilysin. While all ApoE isoforms enhance Ab degradation by neprilysin, ApoE4 appears to have the weakest effect in vitro (Jiang et al., 2008). Extracellularly, the degradation of Ab is mediated by insulin-degrading enzyme (IDE), a protease that is released by microglia and astrocytes into the extracellular milieu and whose levels are reduced by ApoE4 (Jiang et al., 2008; Qiu et al., 1998). The interaction between ApoE and Ab is affected by the lipidation state of ApoE. It has been shown in vitro that lipidated ApoE is more effective at promoting both Ab efflux across the BBB and Ab degradation by proteases (Jiang et al., 2008). Lipidated ApoE is generated in the brain by ATP binding cassette transporter A1 (ABCA1), which transfers cholesterol from the plasma membrane to lipid-poor ApoE (Lawn et al., 1999). Overexpression of ABCA1 increases ApoE lipidation and reduces amyloid deposition in a mouse model of AD (Wahrle et al., 2008); conversely, decreasing ABCA1 expression has the opposite effect (Wahrle et al., 2005). One explanation for this effect is that ApoE binds Ab more efficiently when lipidated (Tokuda et al., 2000); however, ApoE shows minimal interaction with Ab in vivo and may instead affect Ab clearance indirectly (Verghese et al., 2013) (Figure 3). Finally, Ab binding to lipidated ApoE particles can also be enhanced by the acquisition of sulfatides, sulfated derivatives of galactocerebrosides that are primarily produced by oligodendrocytes and form the major components of the myelin sheath Neuron 83, August 20, 2014 ª2014 Elsevier Inc. 777

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Review surrounding the axon (Eckhardt, 2008). Enhanced Ab binding in the presence of sulfatides promotes Ab clearance through ApoE-mediated endocytosis. In AD patients, the sulfatide content in the CNS is reduced, which is mirrored by a reduction of sulfatides in the CSF (Han et al., 2002; Han et al., 2003). This sulfatide loss is specific to AD, as it is not found in other neurodegenerative diseases such as Parkinson’s and dementia with Lewy bodies (Cheng et al., 2003). Lipoprotein receptors play a specific role in this process, as reduction of Lrp1 led to sulfatide loss in a mouse model (Liu et al., 2010). As described earlier, lipoprotein receptors affect both Ab production and clearance. A few key mouse and human studies strongly suggest that clearance is the process primarily affected in AD and by ApoE isoforms (Castellano et al., 2011; Mawuenyega et al., 2010). However, it is also important to note that ApoE4 carriers and preclinical AD patients have hyperactivity in several brain regions and that increased synaptic activity leads to overproduction of Ab (Cirrito et al., 2005; Filippini et al., 2009; Quiroz et al., 2010; for review, see Palop and Mucke, 2010). Whether Ab buildup causes hyperactivity or vice versa in the early phases of disease is still debated. Vascular Ab and Lipoprotein Receptors Cerebral amyloid angiopathy (CAA) is the deposition of amyloid in brain capillaries, leptomeningal vessels, and arterioles. Most commonly, CAA is the result of deposition of Ab in the smooth muscle cell layer, which gradually replaces the smooth muscle wall and leaves the vessel more susceptible to hemorrhage (Vinters, 1987). The majority of patients with AD also have CAA, which can independently trigger intracranial hemorrhage and progressive dementia (Ellis et al., 1996; Rensink et al., 2003). Similar to AD, possession of the ApoE4 allele causes patients to be more susceptible to CAA and hemorrhage, a finding which is mirrored in mouse models of AD (Fryer et al., 2005; Greenberg et al., 1995). Interestingly, ApoE2, which is protective for AD, is associated with an increased risk of hemorrhage when CAA is present (Nicoll et al., 1996). Ab drains out of the CNS through ISF drainage pathways in the smooth muscle cell layer of arterioles, leptomeningal arteries, and capillaries, which is primarily where Ab builds up in CAA. Lrp1 is required for clearance of Ab through smooth muscle cells, since smooth muscle cell-specific knockout of Lrp1 leads to the buildup of both parenchymal amyloid plaques and CAA in a mouse model of AD (Kanekiyo et al., 2012). Smooth muscle cell levels of Lrp1 are reduced in AD (Bell et al., 2009). It is not yet clear if Lrp1 is the only lipoprotein receptor involved in CAA, or if it is the mechanism by which ApoE4 increases CAA risk. For an in-depth review of neurovascular dysfunction in AD, see Zlokovic (2011). ApoE Fragmentation Full-length ApoE may not be the only ApoE form that is involved in AD pathology. In postmortem AD brains, there is an accumulation of C-terminal-truncated ApoE (Huang et al., 2001). While this could simply be the result of increased degradation of ApoE, it has been shown that the addition of ApoE fragments to neuronal cultures leads to the accumulation of NFTs (Brecht et al., 2004). Cell stress in neurons in vitro results in ApoE frag778 Neuron 83, August 20, 2014 ª2014 Elsevier Inc.

mentation and the generation of toxic C-terminal-truncated fragments, which can escape the secretory pathway and cause NFT formation (Brecht et al., 2004). ApoE4 is particularly susceptible to proteolysis due to a domain interaction between Arg-61 and Glu-255, which is facilitated by the Arg-112 specific to apoE4 (Dong and Weisgraber, 1996; Dong et al., 1994), and expression of truncated ApoE4 in transgenic mice leads to AD-like neurodegeneration and death (Harris et al., 2003). These studies, reviewed in Mahley and Huang (2012), indicate a role for ApoE fragments as toxic particles that may cause t aggregation (Brecht et al., 2004), rather than just as markers of ApoE degradation. It is important to point out that ApoE expression is normally absent in neurons but occurs under stress conditions (Xu et al., 2008), and the fragmentation hypothesis requires ApoE to be present in the neuron at the time of injury in order for proteolysis to occur. The Common Thread: ApoE4 and Endocytic Recycling The seemingly disparate effects of ApoE4—altered APP processing, reduced Ab clearance, and impaired synaptic plasticity—share a common mechanism: alterations in endocyctic recycling. To understand this finding, one must remember that the role of lipoprotein receptors and apolipoproteins in the periphery is to transport cholesterol- and triglyceride-rich lipoproteins. In hepatocytes, ApoE-containing particles are taken up primarily through LRP1 and LDLR (Beisiegel et al., 1989; Rohlmann et al., 1998). While cholesterol and ApoB are transported to the lysosome, ApoE recycles back to the surface (Rensen et al., 2000). However, ApoE4 recycles poorly in comparison to the other isoforms, leading to its intracellular accumulation (Heeren et al., 2004). The reason for this may be that at the lower pH that prevails in endosomes, ApoE4 is more likely to form a molten globule (Morrow et al., 2002). The altered structure of ApoE4 at low pH may make it more prone to aggregation, leading to disrupted retroendocytosis. As described in the previous sections, the disruption of endocytic recycling by ApoE4 plays a key role in AD. First, ApoE4 has been reported to promote colocalization of APP and BACE1 in early endosomes, which may lead to increased processing of APP (Rhinn et al., 2013). Second, ApoE4 promotes intraneuronal buildup of Ab by affecting its trafficking (Zhao et al., 2014). Finally, ApoE4 causes reduced surface expression of Reelin receptors, blocking Reelin’s ability to protect the synapse against Ab toxicity (Chen et al., 2010). While the relative contributions of these three mechanisms to AD pathogenesis remain to be determined, these data nevertheless clearly show that trafficking presents an appealing target for AD prevention. Furthermore, a recent genomic study showed that gene expression changes in endocytic trafficking molecules were common to both ApoE4 carriers and LOAD patients (Rhinn et al., 2013). One of the identified trafficking proteins was SV2A, a synaptic vesicle protein. Treatment of human fibroblasts with Levetiracetam, an inhibitor of SV2A, reversed both the endosomal trafficking dysfunction and upregulated APP processing caused by ApoE4 but had no effect in the presence of ApoE3 (Rhinn et al., 2013). Levetiracetam also reversed behavioral and electrophysiological deficits in the hAPP mouse model of AD, and it reversed the abnormal hippocampal hyperactivity found in mild

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Review cognitive impairment (MCI) patients (Bakker et al., 2012; Sanchez et al., 2012). Levetiracetam is currently used clinically for the treatment of epilepsy, where it has several significant side effects, including mood disorders and excessive sleepiness (Bootsma et al., 2007). It is currently unknown if these are on- or offtarget effects, and while inhibiting SV2A with Levetiracetam may not be the ideal therapeutic strategy, taken together with other findings, these results strongly indicate that restoring endocytic recycling is an attractive strategy for preventing AD. Insights and Developments in Diagnostics The abundance of molecular data on the role of ApoE receptors and Ab in AD is now setting the stage for increasingly rational and sophisticated diagnostic and therapeutic approaches. In our clinics today, the prediction, diagnosis, and monitoring of Alzheimer’s disease remains inexact. Drug testing and development occurs either in patients with advanced disease, for whom interventions may be too late, or in a cohort of as yet unaffected individuals, which necessarily then must be very large to achieve statistical significance. Similarly, drug efficacy is primarily determined by cognition scores, which may take months or years to change significantly compared to the placebo control group. It is thus important to develop new ways to monitor disease, and the clinical evaluation of AD is rapidly being transformed by the development of new imaging techniques and biomarkers. Historically, the diagnosis of AD could only be confirmed after death via detection of amyloid plaques and neurofibrillary tangles. Through recent advances in noninvasive imaging technology, amyloid deposition can now be observed in living patients using positron-electron tomography (PET). Patients are injected with Pittsburgh B (PIB) or similar compounds, which are PET tracers that bind to amyloid in the brain (Klunk et al., 2004). Studies of PIB imaging have shown that the PIB signal roughly follows what is already known about amyloid deposition from postmortem tissue. In cognitively normal individuals, no PIB signal is present until around 50 years of age, at which point it starts to appear in a percentage of the population. Once individuals begin to be PIB positive, the amount of signal increases over time (Morris et al., 2009). As the PIB signal crosses a vague threshold, cognitive decline starts to occur, with greater PIB signal predicting greater cognitive loss. Since PIB signal closely shadows amyloid deposition, one can examine risk factors, modifiers, and treatments for AD in living patients. For example, ApoE4 carriers have higher PIB signals at younger ages, while ApoE2 carriers have lower signals (Reiman et al., 2009). The PIB signal also increases at a faster rate in ApoE4 carriers (Grimmer et al., 2010). While PIB PET reveals the static deposition of amyloid, FDGPET and fMRI are able to detect functional loss in the affected areas of brain. FDG-PET measures glucose metabolism in the brain, while fMRI can be used to look at blood flow, oxygen consumption, and network connectivity. These tools, reviewed in Perrin et al. (2009), have shown that changes in connectivity and brain function occur long before plaque deposition and cognitive decline in ApoE4 carriers. Cognitively normal ApoE4 carriers already have decreased regional volume by MRI in those brain areas that are the first to be affected in AD, as well as

decreased glucose metabolism by FDG-PET (Chen et al., 2012). Similarly, young, PIB-negative, ApoE4 carriers already have some fMRI connectivity deficits that are similar to those found in AD patients (Sheline et al., 2010). These functional alterations may begin very early in development, since ApoE4-carrying infants as young as 2 months of age exhibited reduced white matter myelin water fraction (MWF) and gray matter volume (GMV) in several areas affected in AD (Dean et al., 2014). Additionally, ApoE4 carriers show greater hippocampal activation during memory encoding tasks and greater activation of the ‘‘default mode’’ network at rest (Filippini et al., 2009). Since neuronal activity leads to increased levels of Ab, increased hippocampal activity may be both, driver and first indicator of amyloid accumulation in these patients. The availability of these new imaging tools has led to important prospective studies in a large familial AD cohort in Colombia. Individuals in this cohort harbor the E280A mutation of PSEN1, which shifts APP processing toward generation of longer Ab species, particularly Ab42. These individuals follow a temporally fixed path of disease development (Van Vickle et al., 2008). Disease onset occurs within a narrow window around 45 years of age, presenting initially with memory loss and then progressing to include personality changes, loss of language, and finally gait abnormalities (Acosta-Baena et al., 2011; Lopera et al., 1997). Since disease progression is so well characterized in the Colombian cohort, we can study neurological changes that occur before the onset of clinical disease. For example, 15 years prior to disease onset, carriers of the PS1 mutation have increased hippocampal activation despite similar performance during memory tasks relative to healthy controls (Quiroz et al., 2010). The Colombian cohort has already been used to update AD diagnostic criteria in the clinic. Studies in predementia E280A carriers identified new cognition tests that could indicate preclinical dementia, including the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) memory test, the naming of famous faces, and semantic variation (Arango-Lasprilla et al., 2007; Cuetos et al., 2007; Tirado et al., 2008). This year, clinical trials using anti-amyloid therapeutics will begin in predementia individuals of the Colombian cohort. The distinction between ‘‘early-onset’’ AD, found in the Colombian cohort, and ‘‘late-onset’’ AD, found in the majority of patients, has a well-understood mechanistic basis. Both diseases involve elevated levels of Ab, which through mechanisms that are not yet entirely clear triggers tau pathology, cognitive impairment, and ultimately results in neurodegeneration. However, both forms of the same disease, i.e., AD, differ mechanistically in that familial AD involves APP mutations that increase Ab production, whereas ApoE4 impairs clearance of Ab. Moreover, ApoE4 appears to alter brain function early in life (Dean et al., 2014), which may leave certain brain regions more susceptible to Ab toxicity (Figure 4). Alzheimer’s disease is a complex disorder whose underlying molecular and network dysfunctions continue to emerge. It is clear that ApoE4 and lipoprotein receptors have pivotal roles in AD pathogenesis by altering cellular trafficking and Ab clearance on one hand, as well as affecting synaptic plasticity on the other. With so many interacting proteins and higher-order network systems that are affected as the disease progresses, it is vital to take Neuron 83, August 20, 2014 ª2014 Elsevier Inc. 779

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Review Figure 4. ApoE4 and AD Progression

Cognitive Function

E3

E4

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Altered Structure/Function Aβ Deposition Tau Deposition

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Young Adult

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an interdisciplinary approach to reduce this unwieldy complexity to its root causes. We are at a thrilling time in Alzheimer’s research as new molecular insights into disease mechanisms reveal novel neurobiological principles and promising therapeutic approaches to feed the desperately needed drug pipeline. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health, Lupe Murchison Foundation, Ted Nash Longlife Foundation, Consortium for Frontotemporal Dementia Research, Alexander von Humboldt Stiftung, and Brightfocus Foundation. Additionally, we would like to thank Theresa Pohlkamp and Catherine Wasser for their thoughtful reading of manuscript drafts. We thank Nancy Heard and Barbara Dacus for their excellent help with the artwork. REFERENCES Abramov, E., Dolev, I., Fogel, H., Ciccotosto, G.D., Ruff, E., and Slutsky, I. (2009). Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses. Nat. Neurosci. 12, 1567–1576. Acosta-Baena, N., Sepulveda-Falla, D., Lopera-Go´mez, C.M., JaramilloElorza, M.C., Moreno, S., Aguirre-Acevedo, D.C., Saldarriaga, A., and Lopera, F. (2011). Pre-dementia clinical stages in presenilin 1 E280A familial earlyonset Alzheimer’s disease: a retrospective cohort study. Lancet Neurol. 10, 213–220. Akaaboune, M., Allinquant, B., Farza, H., Roy, K., Magoul, R., Fiszman, M., Festoff, B.W., and Hantaı¨, D. (2000). Developmental regulation of amyloid precursor protein at the neuromuscular junction in mouse skeletal muscle. Mol. Cell. Neurosci. 15, 355–367. Andersen, O.M., Yeung, C.H., Vorum, H., Wellner, M., Andreassen, T.K., Erdmann, B., Mueller, E.C., Herz, J., Otto, A., Cooper, T.G., and Willnow, T.E. (2003). Essential role of the apolipoprotein E receptor-2 in sperm development. J. Biol. Chem. 278, 23989–23995.

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ApoE4 carriers have alterations in brain structure and function starting early in life, most notably in AD-related areas. As early as infancy, MRI reveals that there is reduced gray matter volume in posterior/middle cingulate, lateral temporal, and medial occipitotemporal regions in ApoE4 carriers, a trend that continues throughout life (Dean et al., 2014). These findings are mirrored in middle age by reduced glucose metabolism in the same areas in ApoE4 carriers, prior to the onset of cognitive dysfunction (Protas et al., 2013). With age, ApoE4 carriers have earlier deposition of Ab plaques compared to ApoE3 carriers, which is then followed by the development tau tangles and frank atrophy. The altered baseline brain function and accelerated Ab deposition combine to cause ApoE4 carriers to have both an earlier age of disease onset and a more rapid decline in cognitive function (Caselli et al., 2009). Ab, amyloid beta; AD, Alzheimer’s disease; ApoE, apolipoprotein E.

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