Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Neurabin: A Novel Neural Tissue–specific Actin Filament–binding Protein Involved in Neurite Formation

We purified from rat brain a novel actin filament (F-actin)–binding protein of ∼180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue–specific F-actin– binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin–binding domain at the NH₂-terminal region, one PSD-95, DlgA, ZO-1–like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin–cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.During the development of the nervous system, the distal tip of the elongating axon—the growth cone—actively migrates toward its target cell in response to the combined actions of attractive and repulsive guidance molecules in the extracellular environment (Garrity and Zipursky, 1995; Keynes and Cook, 1995; Chiba and Keshishian, 1996; Culotti and Kolodkin, 1996; Friedman and O''Leary, 1996; Tessier-Lavigne and Goodman, 1996). When the growth cone contacts with the target cell, it is transformed into the functional presynaptic terminal (Garrity and Zipursky, 1995; Chiba and Kishishian, 1996). The actin cytoskeleton has been shown to play crucial roles in these processes of the synapse formation (Mitchison and Kirschner, 1988; Smith, 1988; Bentley and O''Connor, 1994; Lin et al., 1994; Mackay et al., 1995; Tanaka and Sabry, 1995).In the developing nervous system, the actin cytoskeleton is prominent in two structural domains of the growth cone, filopodia and lamellipodia (Mitchison and Kirschner, 1988; Smith, 1988; Bentley and O''Connor, 1994; Lin et al., 1994; Mackay et al., 1995; Tanaka and Sabry, 1995). In these domains, actin filament (F-actin)¹ assembled at the leading edge are transported into the center of the growth cone and disassembled there. It has been suggested that this retrograde flow of F-actin is crucial for the growth cone motility. Drugs that disrupt F-actin have also been shown to cause the lamellipodial and filopodial collapse and block the ability of neurons to extend the growth cone in the correct direction (Marsh and Letourneau, 1984; Forscher and Smith, 1988; Bentley and Toroian-Raymond, 1986; Chien et al., 1993). These results suggest that the actin cytoskeleton regulates not only the growth cone motility but also the growth cone directionality. Recently, a variety of guidance molecules and their receptors have been identified (Garrity and Zipursky, 1995; Keynes and Cook, 1995; Chiba and Keshishian, 1996; Culotti and Kolodkin, 1996; Friedman and O''Leary, 1996; Tessier-Lavigne and Goodman, 1996). However, which molecules of the actin cytoskeleton are essential for the growth cone motility and directionality is not well understood.When the growth cone contacts with the target cell, the target cell regulates the development of the presynaptic nerve terminal and the formation of the functional synapse (Bowe and Fallon, 1995; Chiba and Keshishian, 1996). In the established nervous system, the presynaptic and postsynaptic membranes get aligned in space and constitute the synaptic junction (Burns and Augustine, 1995; Garner and Kindler, 1996). Electron microscopic studies have revealed the ultrastructural features of the synaptic junction (Burns and Augustine, 1995; Garner and Kindler, 1996). The presynaptic cytoplasm is characterized by synaptic vesicles (SVs). SVs are not distributed uniformly; SVs cluster together in the vicinity of the presynaptic plasma membrane, where F-actin forms a network and is associated with the presynaptic plasma membrane (Hirokawa et al., 1989). Most SVs within the cluster are linked through thin strands to each other, to F-actin, or to both (Hirokawa et al., 1989). A subset of SVs within the cluster are attached by fine filamentous threads to neurotransmitter release zone at the presynaptic plasma membrane (Hirokawa et al., 1989). The presynaptic submembranous cytoskeleton is assumed to be involved in recruiting Ca²⁺ channels and the components of the SV fusion complex, delivering SVs to the neurotransmitter release zone, and keeping them in place (Burns and Augustine, 1995; Garner and Kindler, 1996). At the inner surface of the post-synaptic plasma membrane, there is an electron dense thickening, called postsynaptic density. The postsynaptic density is assumed to be involved in the selective targeting and accumulation of ion channels and receptors (Burns and Augustine, 1995; Garner and Kindler, 1996). It is also assumed that the presynaptic and postsynaptic submembranous cytoskeleton elements are linked to cell adhesion molecules to regulate the synaptic stabilization and plasticity (Fields and Itoh, 1996; Garner and Kindler, 1996). The presynaptic and postsynaptic submembranous cytoskeleton elements are thought to be composed of spectrin/fodrin, ankyrin, α-adducin, and protein 4.1 isoforms and to be linked to F-actin through these cytoskeleton proteins (Garner and Kindler, 1996). However, little is known about which molecules of the submembranous cytoskeleton are essential for the synaptic transmission and/or the synaptic stabilization.To understand the regulation of the actin cytoskeleton during and after the development of the nervous system, it is of crucial importance to identify F-actin–binding proteins implicated in the synapse formation and function. Therefore, we attempted here to isolate neural tissue–specific F-actin–binding proteins. We isolated a novel neural tissue–specific F-actin–binding protein from rat brain, which may be involved in neurite formation, and named it neurabin (neural tissue–specific F-actin–binding protein).     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Structural Characterization of the Ectodomain of a Disintegrin and Metalloproteinase-22 (ADAM22), a Neural Adhesion Receptor Instead of Metalloproteinase: INSIGHTS ON ADAM FUNCTION?

ADAMs (a disintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signal transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities.The ADAM² family includes over 20 multidomain type I transmembrane glycoproteins that have diverse functions in cell adhesion/signaling and ectodomain shedding of cell-surface receptors or ligands (1, 2). They are broadly implicated in various physiological processes including sperm-egg interactions, development and function of the nervous system (e.g. cell-fate determination, axon guidance, and myelination), immune responses, and embryogenesis (2, 3). Dysregulation of the ADAM family is linked to a wide variety of pathological states including cancer, cardiovascular disease, asthma, Alzheimer disease, and inflammation (3–5). Several ADAMs have been pursued as therapeutic targets (6, 7).ADAMs, together with their phylogenic relatives, the P- III class snake venom metalloproteinases (SVMPs) and ADAMTSs (ADAM with thrombospondin type-1 motif), constitute a subgroup of the metzincin clan of zinc proteinases (8, 9). The extracellular segments of ADAMs contain a prodomain that gets cleaved off during secretion, a metalloproteinase-like domain, a disintegrin domain, and a cysteine-rich domain, which are shared by SVMPs and ADAMTSs, and a unique epidermal growth factor-like domain preceding the transmembrane segment. All ADAMs contain metalloproteinase-like domains, but in humans, only 13 of the 21 members in the family possess the complete zinc binding environment (the HEXGHXXGXXHD sequence motif and the Met turn) in the domain (10). Although these proteolytically active ADAMs can shed cell-surface proteins from the plasma membrane, the other ADAMs are suggested to be non-enzymatic cell adhesion molecules (11, 12). Several ADAMs have been reported to interact with integrins, and the disintegrin-like domains of ADAMs have been suggested for this interaction (13). Despite these suggestions, structural proof that the ADAMs without canonical zinc-binding motif lack enzymatic activities has been absent, and it remains unclear how these molecules are structurally configured to support protein-protein interaction instead of ectodomain shedding.ADAM22 (also named MDC2), one of such postulated non-catalytic ADAMs, was recently identified to serve as the postsynaptic receptor for the secreted neurotransmission modulator LGI-1 at neural synapses (14). The study supports that some ADAMs can function as adhesion molecules rather than metalloproteinases. ADAM22 is predominantly expressed in the nervous systems (15, 16). The Adam22^−/− mice suffered from hypomyelination of peripheral nerves, leading to ataxia, and died before weaning (17). At the synapse, LGI-1 and ADAM22 form a tertiary complex with postsynaptic density-95 (PSD-95), a major scaffolding protein localized to the postsynaptic density of brain synapses, which is associated with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and other signaling proteins (14). In this complex, the extracellular domain of ADAM22 interacts with LGI-1, whereas its cytoplasmic PDZ-binding motif recruits PSD-95. The link of ADAM22 and LGI-1 to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor established their roles in glutamate neurotransmission, consistent with genetic data that all these molecules are associated with epilepsy (17–19). Recently, it was further demonstrated that LGI-1 and LGI-4 bind to ADAM22, ADAM23, and ADAM11 (20).Although ADAMs are functionally important as sheddases or adhesion receptors, the structural information about the ADAM family is limited to only isolated domains, such as the metalloproteinase domains of ADAM17 and ADAM33 and the incomplete disintegrin cysteine-rich domains of ADAM10 (21–23). Their relatives, SVMPs from the snake venom, including VAP-1, VAP-2, and RVV-X (24–26), have revealed a “C”-shaped molecular architecture. These SVMP structures and partial ADAM structures, along with those of the ADAMTS family proteins (27–29), shed light on the general mechanisms of substrate recognition and cleavage by the proteinase-type ADAMs. However, there is little structural information on those non-catalytic ADAMs, which serve as adhesion receptors. In addition, despite a low resolution electron microscopic (EM) analysis of the soluble form of pro-ADAM12, which suggested that the prodomain represents one of the leaves of the four-leaf clover-shaped ADAM12 (30), the structure of a complete ADAM ectodomain, being catalytic or non-catalytic, has been lacking. Here we report the crystal structure of the entire ectodomain of mature ADAM22.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Brain Myosin V Is a Synaptic Vesicle-associated Motor Protein: Evidence for a Ca2+-dependent Interaction with the Synaptobrevin–Synaptophysin Complex

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca²⁺ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca²⁺ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg²⁺. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.Synaptic vesicles are neuronal organelles that sequester, store, and release neurotransmitters. Functionally mature synaptic vesicles are assembled locally, in the nerve terminal, from at least two different types of precursor vesicles (Okada et al., 1995). Once the mature synaptic vesicle becomes docked at the presynaptic membrane, it is prepared (“primed”) to rapidly discharge its shipment of transmitter into the synaptic cleft upon the arrival of an action potential. This secretory response is triggered by the opening of voltage-regulated Ca²⁺ channels and is the principle mechanism used by neurons to exchange information.A majority of the synaptic vesicles in a nerve terminal are sequestered into clusters that are not immediately available for exocytosis (Landis et al., 1988; Hirokawa et al., 1989). Thus, the initial burst of Ca²⁺-triggered exocytosis is presently understood to result from the fusion of predocked vesicles that had matured to a fusion-competent state before the action potential arrived (Südhof, 1995; Rosenmund and Stevens, 1996). The current working model further interprets the existence of vesicle clusters as a potential reserve pool of synaptic vesicles that may be mobilized in a use-dependent manner to replenish the pool of readily releasable vesicles. Several independent lines of experimental evidence support this scheme. Electrophysiological measurements of membrane fusion report that hippocampal synapses recover from complete synaptic fatigue with a time constant of ∼10 s (Stevens and Tsujimoto, 1995; Rosenmund and Stevens, 1996), suggesting that mechanisms exist for actively replenishing the readily releasable pool of synaptic vesicles. The kinetics of synaptic vesicle recycling indicate that recently retrieved vesicle membranes do not completely fulfill the demand for releasable vesicles during periods of intense secretory activity (Ryan et al., 1993; Ryan and Smith, 1995). This analysis implies that a supply of fresh synaptic vesicles must somehow move from a reserve pool to the presynaptic membrane. In fact, movements of synaptic vesicles have now been observed in a variety of different synaptic preparations (Llinas et al., 1989; Koenig et al., 1993; Henkel et al., 1996). These movements are inconsistent with simple diffusion, since they are reported to be both ATP-dependent and vectorial in nature. Integral synaptic vesicle proteins are transported to the terminal by a microtubule-based superfamily of motor proteins, the kinesins (Okada et al., 1995). However, microtubules do not extend into the cortical cytoskeletal matrix of terminals (Landis et al., 1988; Hirokawa et al., 1989), and the kinesins are rapidly degraded upon their arrival in the terminal (Okada et al., 1995). Moreover, recent imaging studies have shown that intracellular particles move along actin bundles in nerve growth cones, rather than microtubules (Evans and Bridgman, 1995). For these reasons, it seems unlikely that the kinesins move synaptic vesicles between their putative storage sites (the vesicle clusters) and the sites of their release (the active zones). Despite intensive study of the molecular events that govern synaptic vesicle recycling, relatively little is known about the mechanisms that control synaptic vesicle movements within the nerve terminal.Brain myosins V (p190, dilute, myr 6) are presently the leading candidates for a synaptic vesicle motor protein (Langford, 1995). In the nervous system, the mouse dilute (Mercer et al., 1991) and chicken myosin V (p190; Espreafico et al., 1992) proteins are localized within neurons and are more abundant than myr 6, which is concentrated in the dentate gyrus and choroid plexus (Zhao et al., 1996). The myosin superfamily includes at least 11 different molecular motors that possess a conserved motor domain attached to a variety of neck and tail domains with distinguishing structural and biochemical characteristics (Cheney et al., 1993b ; Mooseker and Cheney, 1995). Both the conventional myosins II and unconventional myosins V have been localized within presynaptic terminals (Espreafico et al., 1992; Mochida et al., 1994; see also Miller et al., 1992); form a two-headed structure that is capable of generating mechanochemical force and moving actin filaments; and possess actin-activated Mg²⁺-ATPase activity. In addition, both forms of myosins have neck domains that contain variable numbers of putative regulatory calmodulin light chain binding cassettes, and a flexible coiled-coil stalk that is followed by a tail domain which ends as a large (∼400 amino acids) COOH-terminal globular structure (Espreafico et al., 1992; Cheney et al., 1993a ). Diversification of the intracellular localization and functions of the myosins seems to depend on the COOH-terminal tail which is variable in sequence and length (Cheney et al., 1993b ). Myosins II and V appear to regulate complementary presynaptic functions: actin dynamics and the transport of membrane-bound organelles, respectively. The heavy chains of myosins IIA and IIB bind to acidic phospholipids and self-assemble into contractile filaments on the subplasmallemal surface (Murakami et al., 1995; Verkhovsky et al., 1995), which may play an important role in growth cone motility (Miller et al., 1992), the cycling of cortical actin assembly (Bernstein and Bamburg, 1989), and the regulation of neurotransmitter release (Mochida et al., 1994). In contrast, myosins V are not filament-forming, and they colocalize with intracellular membranes (Cheney et al., 1993a ). The class V myosins may have a role in regulating the extension of growth cone filapodia during neuronal development (Wang et al., 1996), as well as the movement of axoplasmic organelles (Kuznetsov et al., 1992; Bearer et al., 1993; Langford, 1995). A potential role for brain myosin V in organelle trafficking and transport is indirectly supported by the finding that this motor protein is capable of moving relatively large (800 nm) artificial beads in motility assays (Wolenski et al., 1995). Moreover, functional null mutations have established that the class V myosins are required in yeast for the intracellular transport of post-Golgi secretory vesicles (Johnston et al., 1991; Lillie and Brown, 1992; Govindan et al., 1995).Myosin motors bound directly to synaptic vesicles may have an important role in regulating the availability of synaptic vesicles for exocytosis. The present study demonstrates that 2,3-butanedione-2-monoxime (BDM),¹ a pharmacological inhibitor of endogenous myosin Mg²⁺-ATPase activity, significantly decreases the Ca²⁺-dependent release of glutamate from isolated nerve endings (synaptosomes). In addition, our data reveal that a major portion of the myosin V in nerve terminals was bound to synaptic vesicles and that this interaction was regulated by Ca²⁺ concentrations that are of physiological relevance. Cross-linking experiments indicated that myosin V specifically complexes with the synaptic vesicle proteins synaptobrevin and synaptophysin. Interactions between these two integral membrane proteins may impose an important additional constraint on the regulated pathway of secretion (Calakos and Scheller, 1994; Edelmann et al., 1995). We now present evidence that the cytoplasmic domain of the synaptobrevin–synaptophysin complex may also function as a binding partner for myosin V, forming a multimeric complex that shall be referred to as the “myosin V transport complex.”     

The Novel Caspase-3 Substrate Gap43 is Involved in AMPA Receptor Endocytosis and Long-Term Depression

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.Synaptic plasticity (the ability of synapses to change in strength) plays an important role in brain development and cognitive function, including learning and memory. N-methyl-d-aspartate receptor (NMDAR)¹-dependent long-term depression of synaptic transmission (LTD) is a major form of synaptic plasticity that leads to long-lasting decreases in synaptic strength. In NMDAR-LTD, synaptic depression is mainly mediated by removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from the postsynaptic membrane through endocytosis (1–3).Caspases are cysteine-dependent proteases that cleave after an aspartate residue (4–6). Primary specificity for aspartate at the cleavage site is so rare in mammalian proteases that only granzyme B, a serine protease derived from lymphocytes, is known to also have such a property (7, 8). Caspases are best known for their pro-apoptotic function in programmed cell death, or apoptosis (9). Caspases activated at the end of a caspase cascade are “effector” caspases, and among them caspase-3 is the dominant one (10). In addition to apoptosis, caspases also play non-apoptotic roles such as in cell differentiation, dendritic development, and memory consolidation (11–14). For instance, our earlier studies show that NMDAR-LTD requires moderate and transient caspase-3 activation, which does not induce cell death (15, 16).In LTD, caspase-3 is activated by the mitochondrial pathway (15, 16). With the opening of NMDA receptors, calcineurin and protein phosphatase 1 are activated to dephosphorylate the Bcl-2 family protein BAD. Dephosphorylated BAD then translocates to the mitochondria, activating BAX, which is also a member of the Bcl-2 family protein. The subsequent release of cytochrome-c from mitochondria leads to the sequential activation of caspase-9 and caspase-3. Active caspase-3 induces AMPA receptor endocytosis, and therefore depression of synaptic strength. The mechanism by which caspase-3 promotes AMPA receptor endocytosis, however, remains to be determined.Caspases'' cellular functions are primarily mediated by the proteolysis of caspase substrates, resulting in change of their functions. Identification of specific proteins cleaved by caspases is therefore key to understanding the mechanisms mediating their biological functions. Several proteomic approaches, developed specifically for this purpose, have led to the identification of more than 2000 caspase cleavage sites (17–22). In particular, the recently developed subtiligase-based method for isolating proteolytic products has led to the identification of >1,000 putative caspase substrates in human samples (21, 23, 24). Subtiligase is an engineered peptide ligase that ligates esterified peptides onto the N termini of proteins or peptides through free α-amines (25). Because the majority of eukaryotic proteins are N-terminally acetylated—and therefore blocked from subtiligase labeling (26)—subtiligase can couple synthetic tagged peptides selectively to the free N-terminal α-amines of proteins derived from proteolysis. These peptide-conjugated proteolytic products can then be affinity-purified, digested with trypsin and sequenced by mass spectrometry. Identification of peptides ligated to the tagged peptide by subtiligase allows researchers to determine cleavage sites within the substrates.In this study, the subtiligase-based proteomic method was used to find capase-3 substrates in rat neurons, resulting in the identification of 81 putative aspartate cleavage sites in 56 proteins. Of these, 37 proteins (human and a single rat orthologs) were not previously reported in the CASBAH database (20), and 13 (human orthologs) are not included in the DegraBase data set compiled using the subtiligase methodology in non-neuronal tissue (21). Using complimentary methods, we further confirmed that, both in vivo and in vitro, caspase-3 cleaves three of these candidate substrates: growth associated protein 43 (Gap43), drebrin (Dbn1), and brain acid soluble protein 1 (BASP1). Surprisingly, we also found that AMPA receptor endocytosis and LTD induction both require caspase-3 to cleave Gap43, a protein well known for its presynaptic functions, at the sites identified by our study.