JOURNAL OF NEUROCHEMISTRY

| 2015 | 132 | 20–31

doi: 10.1111/jnc.12989

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*Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC †Medical College of Acupuncture Moxibustion and Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou, China ‡Department of Neuroscience and Regenerative Medicine, Georgia Regents University, Augusta, GA §Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, School of Life Sciences, East China Normal University, Shanghai, China ¶Department of Neurology, Medical College of Georgia, Georgia Regents University, Augusta, GA **Charlie Norwood VA Medical Center, Augusta, Georgia ††Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia

Abstract Understanding mechanisms governing the trafficking of transmembrane (TM) cargoes to synapses and other specialized membranes in neurons represents a long-standing challenge in cell biology. Investigation of the neuron-enriched endosomal protein of 21 kDa (NEEP21, or NSG1or P21) and Calcyon (Caly, or NSG3) indicates that the emergence of the NEEP21/ Caly/P19 gene family could play a vital role in the success of these mechanisms in vertebrates. The upshot of a sizeable body of work is that the NEEP21 and Caly perform distinct endocytic and recycling functions, which impact (i) a amino-3hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor trafficking at excitatory synapses; (ii) transport to/in neuronal axons; as well as (iii) proteolytic processing of

amyloid precursor protein and neuregulin 1, suggesting roles in neuron development, synaptic function, and neurodegeneration. We argue that their distinct effects on cargo endocytosis and recycling depend on interactions with vesicle trafficking and synaptic scaffolding proteins. As they play complementary, but opposing roles in cargo endocytosis, recycling, and degradation, balancing NEEP21 and Caly expression levels or activity could be important for homeostasis in a variety of signaling pathways, and also lead to a novel therapeutic strategy for disorders like Alzheimer’s disease and schizophrenia. Keywords: Alzheimer’s, clathrin, endocytosis, plasticity, recycling, schizophrenia. J. Neurochem. (2015) 132, 20–31.

The elaborate endosomal system in neurons plays a key role in the morphogenesis and maintenance of specialized domains in cell bodies, axons, and dendrites. Recent studies on trophic factors, ion channels, and neurotransmitter receptors indicate that by sorting and transporting membrane proteins and lipids, endosomal transport is important for neuron survival and connectivity, as well as for synaptic transmission and plasticity (Park et al. 2004; Van der Sluijs

Received September 9, 2014; revised manuscript received October 17, 2014; accepted October 23, 2014. Address correspondence and reprint requests to Clare M. Bergson, Department of Pharmacology and Toxicology, Georgia Regents University, 1459 Laney Walker Boulevard, Augusta, GA 30912 USA. E-mail: [email protected] Abbreviations used: Ab, amyloid b peptide; AMPA, alpha-amino-3hydroxy-5-methylisoxazole-4-propionate; APP, amyloid precursor protein; CME, clathrin mediated endocytosis; EE, early endosome; LE, late endosome; Nrg1, neuregulin 1; RE, recycling endosome; TM, transmembrane.

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‘Function of NEEP21 and Calcyon in Neurons’

and Hoogenraad 2011; Yap and Winckler 2012). For example, the strength of excitatory synapses in the CNS is in large part determined by dynamic use-dependent alterations in glutamatergic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor clathrin-mediated endocytosis (CME) and endosomal recycling (Lin et al. 2000; Carroll et al. 2001; Park et al. 2004; Esteban 2008). In addition, a number of neurodegenerative and psychiatric disorders, including mental retardation, cerebral palsy, Alzheimer’s disease, and schizophrenia, have been linked to dysfunctional endosomal transport (Hirokawa et al. 2010; Gokhale et al. 2012; Rajendran and Annaert 2012; Lamb et al. 2013). In the case of Alzheimer’s disease-associated amyloid b-peptide (Ab)-rich amyloid plaques, Ab peptide production requires convergence of amyloid precursor protein with endosomal proteolytic neurosecretases (Rajendran and Annaert 2012; Buggia-Pr
evot et al. 2013; Das et al. 2013). In general, the molecular framework for underlying the complex endosomal transport system in neurons is not well understood. This review focuses on two closely related, neuron-enriched endosomal proteins, NEEP21 and Caly, and provides an in-depth examination of the multiple lines of evidence indicating that they play complementary roles in synaptic endocytosis and recycling, axonal cargo sorting and transport, as well as neurosecretase substrate proteolytic cleavage. Evolution, protein structure and expression in the CNS The NEEP21/Caly/P19 gene family is characterized by a single TM segment in the middle of the encoded proteins, and a 47 amino acid motif in the C-terminus adjoining the TM domain that is conserved among orthologs (Fig. 1a). Outside of this unique motif, sequence consensus among orthologs is greatest at the N-terminus, and most divergent in the distal C-terminus. Searches of sequence databases suggest this gene family emerged relatively recently, that is, early in vertebrate evolution, with NEEP21 and Caly emerging during different epochs (Muthusamy et al. 2009). Whereas NEEP21 and P19 orthologs are detectable in databases of bony fish, but not urochordate and invertebrate phyla, Caly orthologs are only found in mammalian databases (Muthusamy et al. 2009). Another conserved feature of the NEEP21/Caly/P19 gene family is predominant expression in the CNS, including the apparent family progenitor, zebrafish P19 (Fig. 1b). Accordingly, NEEP21 and P19 were originally identified in cDNA screens for genes expressed in embryonic striatum (SaberanDjoneidi et al. 1995, 1998), about a decade before Caly was identified in a two-hybrid screen of a human brain cDNA library (Lezcano et al. 2000). Northern blot studies show enrichment of NEEP21, Caly, and P19 transcripts in brain and pituitary (Saberan-Djoneidi et al. 1995, 1998; Lezcano et al. 2000). Outside the brain, relatively low levels of Caly transcripts can be detected in kidney, and low levels of both

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Fig. 1 Domain organization of the NEEP21/Caly/P19 family and expression in the CNS. (a) Domain organization of human P19, NEEP21, and Caly. The gray dashed line brackets the 47 residue motif conserved among all family members {L-x-[CG]-x-V-x(0,1)-L-[IV]-[MV]-Y-K-[AV]-x(2)-Y-D-x (2,3)-C-P-[DE]-G-F-[LV]-x-[KR]-x(3)-C-x-P-x-[GST]-L-[DE]-x-Y-Y-[AST][ES]-x-D-[PS]-[ENS]-x-[HR]-[ERS]}. Outside of this unique motif, sequence consensus among orthologs is greatest at the N-terminus, and most divergent in the distal C-terminus (Muthusamy et al. 2009). The region containing the transmembrane (TM) segment is shown in orange, the domains encompassing the GRIP1 and clathrin LC-binding sites in NEEP21 and Caly are outlined in blue and green, respectively. Positions of the AP-1, AP-2, and AP-3 adaptor proteins (Muthusamy et al. 2012), and post-synaptic density (PSD95) (Ha et al. 2012) binding sites are indicated by green and blue vertical lines, respectively. (b) Expression of P19 in the zebrafish (Danio rerio) CNS during embryogenesis. Dorsal view of flat mounted embryo at 48 h postfertilization shows mRNA transcripts in the developing forebrain, midbrain, and hindbrain. Anterior is to the left.

NEEP21 and Caly are present in testes. Yet, neither appears to be expressed in liver or heart, suggesting the NEEP21/ P19/Caly family is not widely expressed in peripheral tissues (Dai and Bergson, 2003). Endosomal and synaptic localization in neurons The subcellular localization of NEEP21 and Caly in neurons has been examined at the light and electron microscopic levels. Localization studies with antibodies or epitope-tagged fusion proteins reveal punctate cytoplasmic staining patterns, presumably reflecting that NEEP21 and Caly are mostly embedded in the limiting membranes of vesicles. This section reviews the specific features of their endosomal distributions and discusses how key differences in localization could underlie the distinct endosomal functions of NEEP21 and Caly in neurons. The majority of NEEP21 is found in the trans-Golgi network (TGN), Rab5-positive early endosomes (EEs), or in mixed Rab5/Rab4-positive sorting endosomes (SEs), whereas much less is detected in Rab11-positive recycling

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endosomes (RE) (Steiner et al. 2002; Hoogenraad et al. 2010). Similarly, Caly is expressed in early endosome antigen 1/Rab5-positive endosomes, in addition to Rab11positive vesicles, and the TGN (NM and CB, unpublished data). Caly can also be detected on the cell surface in unstimulated cells, and the fraction of Caly on the plasma membrane increases within a few minutes of treatments that elevate intracellular calcium (Ca++) levels, or stimulate Ca++ calmodulin II kinase activity (Ali and Bergson 2003). The functional consequences of this dynamic flux of Caly to the cell surface are not yet known, but could be relevant to endosomal mechanisms linked to synaptic plasticity (Henley et al. 2011; Anggono and Huganir 2012; Wang et al. 2012) (see below). In neurons, NEEP21 localizes to vesicular organelles restricted to somatodendritic compartments (Steiner et al. 2002; Utvik et al. 2009). In contrast, Caly antibodies label axonal as well as somatodendritic organelles (Lezcano et al. 2000; Xiao et al. 2006). Within dendritic spines, NEEP21 antibody-labeled gold particles are present in the interior of the spines, and at the base of spines where Rab11-positive endosomes are found (Park et al. 2004; Utvik et al. 2009) (Fig. 2a). Like NEEP21, Caly is associated with endosomes within, and at the base of spines. However, there are also differences in the endosomal localization of NEEP21 and Caly in spines. For example, whereas NEEP21 antibodies prominently label multi-vesicular bodies in spines, Caly antibodies do not. In addition, NEEP21 is enriched subjacent to the post-synaptic membrane specialization called the post-synaptic density (PSD). In contrast, Calylabeled gold particles are found along the walls of spines, just lateral to the PSD (Fig. 2B). The lateral region is referred to as the endocytic zone as several proteins associated with the endocytic machinery localize there including clathrin, adaptor protein 2 (AP-2), and dynamin 2 (Racz et al. 2004). In summary, based on their localization in spines, NEEP21 and Caly seem poised to participate in a variety of endocytic and vesicular trafficking events at excitatory synapses. Interaction with the endocytic machinery Mounting evidence from proteomic studies link NEEP21 and Caly with endosomal trafficking. Moreover, based on the unique sets of interacting proteins discovered, these associations could play a vital role in defining the distinct endocytic and recycling functions of NEEP21 and Caly. In this respect, NEEP21 has been co-purified from the developing brain with syntaxin 13, a t-soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor that regulates recycling of a variety of post-synaptic receptors, including AMPA glutamate receptors in spines (Prekeris et al. 1998; Park et al. 2006). While it is not known whether the association involves direct binding, syntaxin 13 is present on SEs and REs like NEEP21 (Prekeris et al. 1998; Trischler et al. 1999).

Several lines of evidence suggest that the association of NEEP21 and syntaxin13 is dynamically regulated by neuronal activity. For example, syntaxin 13 partitions into the plasma membrane, whereas NEEP21 has not been shown to localize on the cell surface. In addition, the extent of NEEP21/syntaxin 13 co-localization depends on expression of another syntaxin 13 interacting protein, called glutamate receptor-interacting protein 1 (GRIP)-associated protein-1 (GRASP-1), which is expressed in REs. Over-expression of GRASP-1 decreases NEEP21 co-localization with syntaxin 13, suggesting GRASP-1 could compete with NEEP21 for binding syntaxin 13 (Hoogenraad et al. 2010). Although interaction of NEEP21 with syntaxin13 and GRASP-1 and its preferential localization in EEs and SEs associate it with early or intermediate endosomal trafficking and sorting events (Steiner et al. 2002), protein interaction studies first linked Caly with endocytosis (Xiao et al. 2006). Initially, a yeast two-hybrid screen and related approaches revealed that the C-terminal cytoplasmic domain of Caly directly interacts with the neuronal isoforms of clathrin light chain ‘a’ (CLCa), and CLCb. The Caly-binding site maps to the clathrin heavy chainbinding domain in CLC as well as the C-terminus regions of CLC, but not the intervening neuron-specific domain. CLC is generally considered a regulatory subunit for clathrin cage assembly or disassembly, but its actions in cells are not fully understood (Ungewickell and Ungewickell 1991; Ybe et al. 1998; Brodsky 2012; Ferreira et al. 2012). Caly has a stimulatory effect on clathrin assembly both in vitro and in the brain (Xiao et al. 2006). Purified Caly C-terminus increases self-assembly of purified brain clathrin in a dose-dependent fashion; conversely, genetic deletion of Caly (CalyKO) significantly reduces steady-state levels of assembled clathrin. Together, these findings indicate that the Caly/CLC interaction could be an important determinant of clathrin-coated vesicle formation in the brain. The C-terminus of Caly also forms a stable complex with the l subunits of the clathrin adaptor proteins 1, 2, and 3 (AP-1, AP-2, AP-3) indicating Caly could play a more extensive role in vesicle trafficking (Muthusamy et al., 2012). Specifically, AP-1 facilitates transport of cargos from the TGN and REs to the cell surface, while AP-2 plays a role in sequestering cell surface cargo destined for internalization, and AP-3 stimulates movement of cargos from endosomes to late endosomes and lysosome related-secretory organelles (Bonifacino and Traub 2003; Newell-Litwa et al. 2007; Hirst et al. 2011). The AP-binding site involves the YXXΦ motif spanning residues 133-136 in the CLC-binding domain (residues 123–155) in Caly. Mutagenesis of the YXXΦ motif abrogates Caly binding to the ubiquitous and neuronal isoforms of l3, and also lowers binding to l1 and l2 subunits. These data along with additional findings from CalyKO mice (discussed in the ‘Transcytosis and Axonal

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‘Function of NEEP21 and Calcyon in Neurons’

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Fig. 2 Function of NEEP21 and Caly at Excitatory Synapses. Immunogold electron micrographs showing the localization of NEEP21 (Utvik et al. 2009)(a) and Caly (Xiao et al. 2006; Negyessy et al. 2008) (b) in dendritic spines. In (a), NEEP21 and GluR2 antibodies are labeled by 30 mm and 10 mm gold particles, respectively. Arrows in (b) point to Caly-antibody labeling with 1.4 mm gold particles. Note prominent sub- and peri-synaptic localization of NEEP21 and Caly, respectively along with endosomal distribution in the spine interior, and at the base of the spine head. (c) Diagram of an excitatory synapse, highlighting the endocytic, recycling, and degradative pathways activated by NEEP21 and Caly. Both proteins are found in EE’s and recycling endosome (RE’s), and Caly is also expressed on the cell surface, and in lysosomes (Steiner et al. 2002; Ali and Bergson 2003; Xiao et al. 2006). NEEP21 forms a complex with syntaxin 13, and directly interacts with GRIP1/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPAR) binding protein (ABP) (GRIP1) (Steiner et al. 2002, 2005). Knock down (KD) of NEEP21 impacts basal and activated recycling of AMPA receptors leading to lower levels in the synapse, and preferential sorting to lysosomes. GRIP1 dephosphor-

ylation is associated with AMPA endocytosis, and interaction with NEEP21. Subsequent phosphorylation of GRIP1 by an NEEP21associated kinase destabilizes the NEEP21/GRIP1 complex, which presumably facilitates recycling of GluR2 to the synapse (Kulangara et al. 2007). Caly directly interacts with the l subunits of AP-1, AP-2, AP-3, clathrin light chain (CLC), post-synaptic density (PSD-95), and the C-terminal cytoplasmic segments of GluR1, R2 and R3 subunits (Xiao et al. 2006; Vazdarjanova et al. 2011; Ha et al. 2012; Muthusamy et al. 2012). Deletion of Caly (CalKO) inhibits stimulated internalization of GluR1 and GluR2/3, while constitutive AMPA receptor endocytosis and recycling is unaffected. The Caly CLCbinding domain (GST-Cal123–155) blocks reduction in AMPA excitatory post-synaptic current (EPSCs) following LTD stimulation (Davidson et al. 2009). The effects of CalyKO on GluR internalization appear to be independent of GluR subunit sorting to recycling or lysosomal pathways. However, Caly over-expression (CalOE) is associated with a reduction in total levels of GluR subunits (Vazdarjanova et al. 2011). Images in panel a, b used with permission from Elsevier and the author (C.B.), respectively.

Transport’ section below) implicate Caly in endosomal transport from a variety of intracellular membranes, as well as from the cell surface (Muthusamy et al. 2012).

Role in endocytosis and recycling The importance of Caly and NEEP21 in vesicle trafficking was first demonstrated for transferrin (Tfn), a well-studied

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housekeeping cargo internalized by CME. Tfn recycling has been extensively studied in heterologous cells, and modeling of the data suggests Tfn recycling involves two kinetically distinct routes, with recycling via Rab5/Rab4-positive SEs predominating over slower recycling via Rab4/11-positive REs (Sheff et al. 1999; Trischler et al. 1999). Supporting this model, the PI3-kinase inhibitor wortmannin (WRT) produces enlarged Rab5/Rab4-positive vesicles, which accumulate internalized Tfn, resulting in a major delay in Tfn recycling. In contrast, the guanine-nucleotide exchange factor inhibitor brefeldin A (BFA) increases tubulation of Rab4/Rab11-positive REs, in which Tfn does not accumulate to a great extent. Thus, BFA treatment results in only a minor delay in Tfn recycling (Klausner et al. 1992; S€onnichsen et al. 2000). Tfn localizes to somatodendritic regions in neurons (West et al. 1997). The role of NEEP21 and Caly in Tfn uptake and recycling has been examined in a variety of cell systems, including neurons, and complementary roles have been described. NEEP21 knock down by anti-sense (NEEP21-AS) or over-expression (OE) alters Tfn recycling (Steiner et al. 2002). In contrast, Caly-KO and -OE alter Tfn internalization, but not recycling (Xiao et al. 2006). NEEP21-AS delays recycling of internalized Tfn, whereas NEEP21-OE increases both internalization and recycling, without affecting total TfnR levels. Further, studies in PC12 cells show that NEEP21 antibody labeling co-localizes with internalized Tfn in WRT-treated, but not BFA-treated cells (Steiner et al. 2002). Taken together, Hirling’s group proposed that the effect on uptake seen in NEEP21-OE cells could be secondary to major effect in promoting Tfn recycling from an early or intermediate recycling compartment (Steiner et al., 2002). In contrast, Caly-OE enhances the rate of TfnR endocytosis by nearly two-fold, whereas rates of internalized Tfn depletion are indistinguishable from controls. Likewise, deficits in Tfn uptake, but not recycling are detected in neocortical neurons from CalyKO mice (Xiao et al. 2006). Based on biochemical data described in the preceding section, the mechanism could well involve Caly’s stimulatory effect on clathrin assembly. Role in GPCR endocytosis and recycling NEEP21 and Caly also regulate the endosomal trafficking of G-protein-coupled receptors (GPCRs). Within the CNS, endocytosis and recycling of GPCRs plays an important role in signaling via a wide range of neurotransmitter and neuromodulatory systems (Hanyaloglu and von Zastrow 2008). The role of NEEP21 in GPCR trafficking has been studied in the context of the receptors for the 13 amino acid neuropeptide neurotensin (NTSR1, NTSR2), whereas Caly has been examined with respect to the dopamine D1 receptor (D1R). The data thus far indicate that the distinct vesicle trafficking functions of NEEP21 and Caly described above for Tfn also apply to GPCRs.

NTS1R is a ‘non-recycling’ receptor, meaning that upon internalization, it sorts from EEs to a degradative pathway leading to lysosomes. In contrast, NTS2R recycles from endosomes via both the WRT- sensitive and BFA-sensitive pathways. Studies conducted in Cos-7 cells, which express low levels of NEEP21, provide a mechanism to reconcile the apparently default sorting of internalized NTS1R to lysosomes (Debaigt et al. 2004). Recycling rather than degradation is observed in Cos-7 cells that over-express NEEP21 (NEEP21-OE), suggesting that NTS1R can enter the recycling pathway if levels of NEEP21 are sufficiently high. Also, in Cos-7 cells expressing NEEP21-AS, NTS1R levels are down-regulated to a much greater extent by NTS treatment compared to effects on untransfected or NEEP21OE cells, indicating that the degradation of the receptor is augmented when NEEP21 levels are diminished. Presumably, NEEP21-OE inhibits NTS1R degradation by diverting internalized receptors into a WRT-sensitive recycling pathway, analogous to the effects of NEEP21-OE on Tfn uptake and recycling. Additionally, as with the Tfn studies, neither NEEP21-AS nor NEEP21-OE alters rates of NTSR1 internalization, again arguing against a role for NEEP21 in receptor internalization. Instead, both sets of data reinforce the idea that NEEP21 accelerates receptor recycling from an early or intermediate endosomal compartment. Co-localization studies in brain as well as co-immunoprecipitation studies in HEK293 cells suggest that Caly and D1Rs form a protein complex (Lezcano et al. 2000, 2006). More recent work suggests that in neurons, the complex could also include post-synaptic density 95/disc large/zona occludens-1 (PDZ)-domain protein (PSD-95), which is an abundant molecular scaffold protein at excitatory synapses (Ha et al. 2012). This finding is intriguing, given the perisynaptic localization of Caly and D1Rs in dendritic spines. Formation of the complex involves direct interaction between the C-terminal most four amino acids glutamine serine proline lysine (QSPK) of Caly and the PDZ repeat 1 (PDZ1) in PSD-95. In addition, interaction with PSD-95 is stabilized by phosphorylation of Caly serine residue 169 following stimulation of D1Rs or protein kinase C (PKC). Importantly, alanine mutagenesis of serine 169 inhibits PKCas well as agonist-stimulated removal of surface D1Rs, suggesting that phosphorylation of this residue is critical for D1R endocytosis. However, details of how PSD-95 impacts D1R internalization are currently unclear. Although the effects of Caly expression alone on D1R internalization were not examined in this study (Ha et al. 2012), previous work indicates that even in the absence of PSD-95, the Caly/D1R complex is stabilized by PKC activation (Lezcano et al. 2000), and that Caly stimulates robust internalization of D1Rs (Xiao and Bergson, SFN abstract 2005). Together, these studies support the idea that Caly stimulates receptor internalization, which is in agreement with the effects of Caly-OE and Caly-KO on Tfn uptake.

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Role in AMPA receptor endocytosis and recycling The localization of NEEP21 and Caly in dendritic spines, coupled with their well-established role in vesicular trafficking, begs the question of whether NEEP21 and Caly could regulate excitatory synaptic transmission, and synaptic plasticity. The primary mediators of excitatory synaptic transmission in brain are AMPA glutamate receptors. AMPA receptors are ligand-gated ion channels assembled as heteromeric tetramers from a combination of four different subunits (GluR1-GluR4). In adult hippocampus, the receptors comprised mainly of GluR1/R2 or GluR2/R3 subunits (Wenthold et al., 1996). Notably, the GluR2/3 subunits constitutively cycle in and out of the synapse via the endosomal recycling system. They are also inserted and removed from synapses in an activity-regulated, or stimulated fashion (Luscher et al. 1999; Noel et al. 1999; Lin et al. 2000; Shi et al. 2001). CME is a key step in both the constitutive cycling and the stimulated removal of synaptic AMPA receptors (Carroll et al., 1999b; Man et al., 2000; Wang and Linden 2000). Stimulated GluR subunit trafficking plays a role in two paradigms of synaptic plasticity, longterm synaptic potentiation (LTP) and long-term depression (LTD) which result in an enduring (> 45 min) increase and decrease, respectively, in AMPA current amplitude, presumably reflecting a corresponding change in synaptic AMPA receptor number. While CME and endosomal recycling clearly plays an important role in AMPA receptor-dependent synaptic plasticity (Lin et al. 2000; Carroll et al. 2001; Park et al. 2004; Esteban 2008), the configuration of the vesicle trafficking machinery at excitatory synapses is not well understood. Investigations of NEEP21-KD and CalyKO in hippocampal neurons provide support for a role of NEEP21 and Caly in regulation of AMPA receptor vesicular trafficking and synaptic plasticity (Fig. 2c). Interestingly, the effects of NEEP21 KD and CalyKO appear to be selective for postsynaptic responses, as neither strategy alters fundamental electrophysiological properties of CA1 neurons in hippocampus, nor pre-synaptic function (Alberi et al. 2005; Davidson et al. 2009). Knock down by NEEP21-AS in hippocampal neurons exerts pleiotropic effects on both basal and evoked transmission. At baseline, NEEP21-AS transfected neurons exhibit a low AMPA to NMDA current ratio presumably due to lower levels of synaptic AMPA-mediated excitatory post-synaptic current (EPSC). Further, AMPA current amplitude does not increase in NEEP21-AS transfected neurons following inhibition of CME, indicating that NEEP21 plays a role in constitutive recycling. NEEP21-AS also impacts AMPA-receptor-dependent synaptic plasticity. While LTD producing stimuli produce greater levels of depression in the NEEP21 AS transfected cells, long-term synaptic potentiation inducing stimuli fail to evoke a prolonged increase of AMPA excitatory post-synaptic current (EPSC). In addition, NEEP21-AS reduces GluR1

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and GluR2 recycling to the surface following stimulation of recycling. Additionally, in response to stimuli that promote recycling of GluR2 subunits, as opposed to sorting to lysosomes, increase co-localization of internalized GluR2 subunits with endogenous NEEP21. Altogether, these effects of NEEP21-AS suggest NEEP21 promotes both constitutive and activity-dependent GluR2 recycling. In contrast to NEEP21, Caly deletion (CalyKO) impacts stimulated, but not basal AMPA-mediated transmission (Davidson et al. 2009). At baseline, the AMPA/NMDA ratio as well as AMPA and NMDA-dependent current/ voltage relationships detected in CalyKO neurons are indistinguishable from wild type. As mentioned above, AMPA receptor internalization is a critical step in LTD, which can be evoked in CA1 hippocampal neurons by low frequency of Schaffer Collaterals. Alternatively, chemical LTD (cLTD) can be evoked in neurons in primary culture by a brief NMDA application (Carroll et al., 1999b; Man et al., 2000; Wang and Linden 2000). Both protocols fail to evoke long-term reduction in EPSC amplitude in the CalyKO samples, but do so in wild-type neurons. However, transfection of green fluorescent protein (GFP)-tagged Caly into CalyKO neurons rescues the cLTD response, suggesting that Caly is necessary and sufficient for AMPA-dependent LTD. While levels of cell surface GluR1 and GluR2/R3 subunits are equivalent in CalyKO and wild-type neurons, immunolocalization studies support the idea that the CalyKO LTD phenotype involves deficits in receptor endocytosis. Stimulated internalization of GluR1 and GluR2/3 subunits is reduced 2–3 fold in CalyKO neurons compared with levels detected in wild-type neurons. In addition, the effects of deleting Caly on GluR subunit internalization are independent of whether the stimulation protocol promotes sorting to lysosomes, or recycling to the plasma membrane (Ehlers 2000; Lin et al. 2000; Lee et al. 2004). Therefore, Caly appears to act upstream of the recycling/degradation decision (Lakadamyali et al. 2006). While a definitive mechanism for how Caly regulates GluR endocytosis is not established, it could involve direct interaction as pull-down studies show that fusion proteins containing the Caly and GluR C-termini co-precipitate (Vazdarjanova et al. 2011). Interaction with synaptic scaffolding proteins Evidence has emerged for involvement of the synaptic scaffold and endocytic machinery in the mechanisms by which NEEP21 and Caly regulate AMPA receptor trafficking and synaptic plasticity (Steiner et al. 2005; Kulangara et al. 2007; Davidson et al. 2009). Specifically, amino acids 129– 164 in the NEEP21 C-terminus were found to bind to the glutamate receptor AMPAR-binding protein-interacting protein1 (GRIP1) (Fig. 1a). GRIP1 is a type II PDZ protein that localizes to the plasma membrane and intracellular compartments in dendritic spines, and also interacts with GluR2 and GluR3. NEEP21 binds to the C-terminal domain PDZ repeat

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7 in GRIP1, while GluR2 interacts with PDZ repeats 4 and 5. Several lines of evidence link the NEEP21/GRIP1 interaction in GluR2 recycling via formation of a tripartite complex. For example, treatments that stimulate AMPA receptor recycling increases levels of GRIP1 and GluR2 co-precipitating with NEEP21, whereas lower levels of the complex co-precipitate following treatments that promote sorting to degradative pathways. Studies with the GRIP1 interaction domain of NEEP21 suggest that the interaction between NEEP21 and GRIP1 plays a significant role in GluR2 recycling, as well as in diverting GluR2 away from pathways linked to lysosomes (Kulangara et al. 2007; Hirling 2009). For example, competitive inhibition of the interaction by transfection of the NEEP21 GRIP1-binding domain thwarts GluR2 recycling. Consistent with this idea, lower levels of surface GluR2 are detected in GFP-NEEP21-129-164 transfected neuron both at baseline, and after treatments that stimulate recycling. In addition, GluR2 accumulates in early endosome antigen 1 (EEA1)-positive and lysosome associated membrane protein 1 (LAMP1)-positive vesicles in cells expressing GFP-NEEP21-129-164. Further, the effects of NEEP21 interaction appear to be selective for GluR2 as neither surface expression nor stimulated recycling of GluR1 are impacted by GFP-NEEP21-129-164. The current data fit with a model, whereby GRIP1 is phosphorylated when associated with the synapse, and dephosphorylated when associated with GluR2 in endosomes. Hirling’s group found that a kinase that co-precipitates with NEEP21 phosphorylates GRIP1 (Kulangara et al. 2007). While the phosphorylating kinase is not known, its activity is stimulated by AMPA or NMDA treatment, which results in increased levels of GRIP1 phosphorylation at serine residue 917. Dephosphorylation of serine 917 facilitates interaction of GRIP1 with NEEP21 and entrance of GluR2 into the recycling pathway. In contrast, rephosphorylation of GRIP1 by the NEEP21-associated kinase destabilizes the NEEP21:GRIP1 complex, and presumably promotes transfer of GluR2 back to the synapse. The findings of Kulangara et al., (2007) argue that GRIP1 interaction with NEEP21 is necessary for GluR2 subunit progression along a recycling pathway. They also indicate that the NEEP21:GRIP1 complex is dynamically regulated in an interdependent fashion by neuronal stimulation and phosphorylation. Involvement of Caly interaction with CLC in the stimulated removal of AMPARs from synapses is supported by electrophysiologial studies in brain slices. Specifically, a peptide containing the Caly CLC-binding domain (GSTCal123–155) perfused into wild-type CA1 neurons via the patch pipette effectively blocks the ability of low frequency stimulation to reduce AMPA EPSC amplitude. In contrast, perfusion with GST protein only is ineffective in blocking the response. The mechanism by which Cal123-155 blocks LTD is not known but could involve inhibition of clathrin-

coated vesicle formation. It is interesting to note that stimulated but not constitutive AMPAR endocytosis is also impaired in knockouts of another CLC-interacting protein, huntingtin interacting protein 1 (Metzler et al. 2001). Like Caly, huntingtin-interacting protein 1 stimulates clathrin assembly in vitro (Chen and Brodsky 2005; Xiao et al. 2006). An unresolved issue is why deletion of these CLCinteracting proteins manifest as defects in stimulated endocytosis of AMPA receptors, whereas alterations in clathrin heavy chain interacting proteins, for example, AP2 or Arc/Arg3.1, also impact constitutive internalization of these receptors (Chowdhury et al. 2006; Kastning et al. 2007). Roles in transcytosis and axonal transport In addition to regulating the trafficking of synaptic receptors, NEEP21 and Caly both play a role in sorting and axonal accumulation of cargos originating in somatodendritic, Tfn-positive endosomes. The acccumulation of membrane proteins in axons can occur via a variety of mechanisms, including specific targeting to axons immediately following synthesis. In the case of newly synthesized proteins non-selectively sorted to both axons and dendrites, at least two other mechanisms are known to support axonal accumulation. These mechanisms involve either selective degradation in dendrites, or directed trafficking from dendrites to axons. The latter mechanism appears to be akin to the transcytotic pathway in polarized epithelial cells, involving trafficking of apical domain proteins that are initially sorted to the basolateral domain (Von Bartheld 2004). An alternate pathway involves the AP-3 adaptor protein sorting and targeting of dendritic cargos to axons (Newell-Litwa et al. 2007). In this section, we present evidence indicating that NEEP21 and Caly regulate axonal cargo sorting and accumulation, albeit via different mechanisms. A role for NEEP21 in transcytosis is based on studies of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCam), which is transported from endosomes in somatodendritic regions to axons (Yap et al. 2008) (Fig. 3a). L1/NgCam is typically enriched on the axonal surface; however, Yap et al. (2008) found that substantial amounts of L1/NgCam accumulate on the surface of soma and dendrites in NEEP21-AS transfected neurons. Live cell imaging studies with GFP-tagged NEEP21 further revealed that L1/ NgCam transiently co-localizes with NEEP21 following internalization into Tfn-positive endosomes in neuronal dendrites. The L1/NgCam/NEEP21-positive endosomes appear to be large, non-motile way stations for L1/NgCam prior to transfer to motile endosomes that are NEEP21 and Tfn-negative. Not only does NEEP21-AS transfection result in less accumulation of L1/NgCam in axons, it is also associated with more endocytosed L1/NgCam in EEs and lysosomes. Therefore, the data suggest that as with GluR and

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(a)

Fig. 3 Roles of NEEP21 and Calcyon in Transcytosis and Axonal Transport. (a) L1/ NgCam is enriched on the axonal surface as a result of endosomal transport from the somatodendritic domain (transcytosis). In dendrites, L1/NgCam co-localizes with NEEP21 (blue line) following internalization into Tfn- positive endosomes. L1/NgCam/ NEEP21-positive endosomes tend to be large and non-motile. Subsequently, L1/ NgCam is transported in the retrograde direction via motile endosomal transport carriers (TC) that are NEEP21 and Tfnnegative. Knock down (KD) of NEEP21 results in more L1/NgCam accumulation in EE’s and lysosomes, and less in axons (Yap et al. 2008). (b) AP-3 facilitates sorting of some synaptic cargos (e.g., ZnT3) from Tfn-positive endosomes in the somatodendritic compartment to synaptic-like microvesicles (SLMVs), which in neurons are found in axon terminals (Salazar et al. 2004). Both membrane recruitment of AP-3 and targeting of AP-3 cargos are significantly impaired by deletion of Caly (CalyKO), as is transport of ZnT3 to axon terminals (Muthusamy et al. 2012).

(b)

NTS1R, NEEP21 plays a decisive role in sorting internalized transcytotic cargo to motile transport carriers destined for axons, essentially by diverting cargos away from a degradative pathway. The discovery that Caly-regulated axonal cargo transport stemmed from investigation of its interaction with the l3 subunit of the AP-3 (Muthusamy et al. 2012) (Fig. 3b). AP-3 is involved in sorting cargos from endosomes to lysosomes and lysosome-related organelles in all cell types. In neuroendocrine cells, AP-3 promotes trafficking of cargos from Tfn-positive endosomes to synaptic-like microvesicles (Newell-Litwa et al. 2007). Likewise, in neurons, AP-3 increases sorting of cargos from somatodendritic Tfn-positive endosomes to axons (Danglot and Galli 2007; Newell-Litwa et al. 2007). Membrane recruitment of AP-3 in brain is significantly impaired by deletion of Caly (Muthusamy et al. 2012). In addition, targeting of two AP-3 cargos: zinc transporter 3 (ZnT3) and phosphoinositol-4-phosphate kinase IIa (PI4KIIa) (Salazar et al. 2004, 2005; Seong et al. 2005; Craige et al. 2008) is

reduced in mossy fibers of the hilus of the dentate gyrus in CalyKO mice. Roles in amyloid precursor protein (APP) and type I neuregulin1 processing Recent studies indicate NEEP21 and Caly could be important for proteolytic cleavage of amyloid precursor protein (APP) and type I neuregulin1 (Nrg1). APP and type I Nrg1 are type I TM proteins, and both are substrates of b secretase 1 (BACE1), a membrane-bound aspartyl protease with a pH optimum of 4.5 (Fleck et al. 2012; Haass et al. 2012). BACE1 cleavage in the extracellular N-terminal domain (ED) of APP is a key step in production of Ab-peptide, a molecule that has been implicated in the etiology of Alzheimer’s disease. Similarly, type I Nrg1 is a leading candidate gene for schizophrenia (Mei and Xiong 2008; Shamir et al. 2012; Mei and Nave 2014), BACE1 cleavage is a critical step in generating the biologically active form of this trophic factor (Hu et al. 2006, 2008; Savonenko et al. 2008; Luo et al. 2011, 2013).

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Vesicle trafficking appears to be a key aspect of the mechanisms involved in Ab production, and type I Nrg1 ED shedding. For example, several lines of evidence suggest that generation of Ab peptide from APP requires CME, and convergence with BACE1 in acidic endosomes (Sannerud et al. 2011; Rajendran and Annaert 2012; Zhu et al. 2012; Buggia-Pr
evot et al. 2013; Chia et al. 2013; Das et al. 2013; Kandalepas et al. 2013). Similarly, early studies of the neuromuscular form of type I Nrg1 (i.e., ARIA) suggest that soluble type I ED is released from a pool of precursor internalized from the plasma membrane, and that ED shedding is inhibited by an increase in endosomal pH (Loeb et al. 1998). BACE1 is also a type I TM protein, and itself shuttles between the cell surface and endosomes after synthesis and maturation in the Golgi (Sannerud et al. 2011; Prabhu et al. 2012; Chia et al. 2013; Das et al. 2013). Here, we discuss recent studies with APP and type I Nrg1 indicating that NEEP21 tempers, whereas Caly potentiates processing of BACE1 substrates. We argue that differences in the inherent endosomal trafficking functions of NEEP21 and Caly could underlie these opposing effects. Studies from the Sisodia laboratory indicate that NEEP21 regulates Ab production. Specifically, NEEP21-OE appears to inhibit Ab generation (Norstrom et al. 2010). Using the Swedish/Nordic isoform of APP (APPswe), which is more efficiently cleaved by BACE1, Norstrom et al. detected lower levels of Ab in the media of NEEP21 transfected cells. Although these studies were conducted in the N2a neuroblastoma cell line, there is evidence to suggest NEEP21 could also regulate APP processing in brain as NEEP21 copurifies with APP from transgenic mouse brain. The mechanism by which NEEP21 interferes with APP processing has not been worked out. Yet, given the role of NEEP21 in cargo recycling, it is tempting to speculate that stimulated recycling as the result of NEEP21-OE lessens endosomal convergence of APP and BACE1. Recent work from our laboratories revealed that Caly stimulates BACE1 processing of type I Nrg1 (Yin et al. in press). Levels of type I ED shed into the extracellular media are dramatically increased when Caly is co-transfected, and levels of cleaved type I Nrg1 C-terminus in lysates are also elevated. Furthermore, the mechanism depends on CME and was blocked by expression of the CalyATEA mutant defective in binding adaptor proteins. Intriguingly, Caly appears to exert similar effects on Nrg1 processing in brain, as levels of cleaved Nrg1 C-terminus are elevated in hippocampus of Caly over-expressing (CalOE) mice, whereas lower levels are detected in CalKO mice. In the CNS, Nrg1 ED binds ErbB tyrosine kinase receptors and activates a signaling cascade, which stimulates release of gamma-aminobutyric acid (GABA) from interneurons. Consistent with the idea that Caly stimulates Nrg1 maturation and ED shedding in brain, GABA transmission is elevated in hippocampal neurons of CalyOE mice, as are levels of ErbB4

phosphorylation, whereas the opposite scenario holds for CalKO mice. ErbB kinase inhibition blocks the CalyOEdriven GABAergic synaptic alterations, supporting the idea that enhanced Nrg1 signaling is involved. As in the case of NEEP21 inhibiting Ab production, the mechanism by which Caly stimulates type I Nrg1 maturation and ED release is not fully understood. Given the ability of Caly to stimulate cargo trafficking to endosomes with low lumenal pH (e.g., EE’s and late endosomes) (Xiao et al. 2006; Muthusamy et al. 2012), Caly could promote either CME or AP-3 dependent targeting of type 1 Nrg1 and/or BACE1. Further studies are needed to validate this hypothesis, as well as to understand whether the alterations detected in NEEP21-OE and Caly-OE cells apply to a range of BACE1 substrates, and/or perhaps to BACE1 itself. Perspectives for future research This review provides an integrative overview of recent data on the function of the closely related vertebrate-specific proteins NEEP21 and Caly in the CNS. The evidence in large part supports a model whereby NEEP21 and Caly carry out unique but complementary vesicle trafficking functions that impact cell biological processes critical for CNS development, learning and memory, and neurodegeneration. These functions range from trafficking of synaptic receptors, transcytosis, and transport of axonal cargoes, to regulating the proteolytic processing of BACE1 substrates associated with brain development and disease. Despite making substantial progress in understanding the function of this recently evolved gene family, several important questions remain to be explored. Among these is whether P19, the putative NEEP21/Caly/P19 gene family progenitor, regulates endocytosis, recycling, or transcytosis. Information on the vesicle trafficking functions and cargoes impacted by P19 should provide a clearer understanding of factors driving the emergence of the NEEP21/Caly/P19 gene family in vertebrates. Another intriguing question is whether Caly and NEEP21 form a protein complex that regulates movement of cargoes in a coordinated fashion. If so, it would be important to learn what signaling pathways dictate the dynamics of complex formation, and how this affects cargo sorting. Finally, could NEEP21/Caly/P19 gene family members serve as therapeutic targets for neurodegenerative and psychiatric disorders? Indeed, future research on this gene family will not only further our understanding of vesicle trafficking mechanisms underlying optimal synaptic transmission in vertebrates, but could also have implications for therapeutic strategies exploiting the roles of NEEP21 and Caly in these mechanisms.

Conclusions The closely related neuron-enriched proteins NEEP21 and Caly interact with distinct elements of the endosomal and

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synaptic scaffolding machinery. Electrophysiological and cell biological studies indicate that their integration with the vesicle trafficking system is required for synaptic plasticity, as well as for transcytosis and targeting of axonal cargos. Finally, NEEP21 and Caly exert opposing effects on the proteolytic cleavage of amyloid precursor protein and type I neuregulin1. As they play complementary roles in cargo endocytosis, recycling, and degradation, balancing NEEP21 and Caly expression levels or activity could be important for homeostasis in a variety of signaling pathways, and also lead to a novel therapeutic strategy for disorders like Alzheimer’s disease and schizophrenia.

Acknowledgments and conflict of interest disclosure We thank Drs. Lynn D Selemon (Yale University) and Bettina Winckler (Univ of Virginia) for their critical comments on an earlier version of the manuscript. The authors thank the following funding agencies for their support: Georgia Regents University; NIH; NARSAD. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

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‘Function of NEEP21 and Calcyon in Neurons’

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© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 20--31