A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching.

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1-sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Effect of genetic deletion of the vanilloid receptor TRPV1 on the expression of Substance P in sensory neurons of mice with adjuvant-induced arthritis.

The neuropeptide Substance P (SP), expressed by nociceptive sensory afferents in joints, plays an important role in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release SP from their central and peripheral axons, suggesting a functional link between SP and the capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1). The expression of both TRPV1 and SP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release SP is not completely understood. We here wanted to ascertain whether the increase in the number of SP-positive primary afferents in arthritis may be affected by genetic deletion of TRPV1. For this, we used immunohistochemistry to quantify the expression of SP in primary afferent neurons in wild-type mice (WT) vs. TRPV1-knockout (KO) mice with adjuvant-induced arthritis (AIA). We found that the expression of SP in DRG (1) increased significantly over naïve level in both WT and KO mice 3 weeks after AIA, (2) was significantly higher in KO mice than in WT mice in naïve mice and 2-3 weeks after AIA, (3) was significantly higher on the side of AIA than on the contralateral, vehicle-injected side at all time points in WT mice, but not in KO mice, and (4) increased predominantly in small-size neurons in KO mice and in small- and medium-size neurons in WT mice. Since the size distribution of SP-positive DRG neurons in arthritic TRPV1-KO mice was not significantly different from that in naïve mice, we speculate that the increased expression of SP is unlikely to reflect recruitment of A-fiber primary afferents and that the higher expression of SP in KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit.

Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain.

Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (glial fibrillary acidic protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (adjuvant-induced arthritis, AIA), and neuropathic pain (partial sciatic nerve ligation, PSNL). We found that (i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naive WT mice; (ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7-14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7-14 days after AIA, and 7 days after PSNL; and iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14-21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain.

Increased expression of CGRP in sensory afferents of arthritic mice--effect of genetic deletion of the vanilloid receptor TRPV1.

The neuropeptide calcitonin gene-related peptide (CGRP), expressed by nociceptive sensory afferents in joints, is an important mediator in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release CGRP from their central and/or peripheral axons, suggesting a functional link between CGRP and the capsaicin receptor TRPV1. The expression of both TRPV1 and CGRP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release CGRP remains unclear. We here wanted to ascertain whether the increase in the number of CGRP-positive primary afferents during arthritis may be affected by genetic deletion of TRPV1. For this, we quantified the expression of CGRP in primary afferent neurons in DRG in wild type mice (WT) vs. TRPV1-KO mice with adjuvant-induced arthritis (AIA), using immunohistochemistry. We found that the fraction of DRG neurons that were immunopositive for CGRP (1) was higher in naïve TRPV1-KO mice than in naïve WT mice, (2) increased progressively 3-21 days after induction of AIA, and (3) this increase was bilateral but significantly greater on the complete Freund's adjuvant-injected side than on the incomplete Freund's adjuvant-injected side in TRPV1-KO mice. The increased expression of CGRP in AIA may reflect a phenotypic switch of primary afferents from non-peptidergic to peptidergic and the larger increase in TRPV1-KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit.

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat.

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.

Vanilloid receptor TRPV1-positive sensory afferents in the mouse ankle and knee joints.

TRPV1, a cation channel on sensory nerves sensitive to heat and capsaicin, plays an important role in the transduction of noxious stimuli to the spinal cord. It is expressed by neurons in dorsal root ganglia (DRG) that may also express neuropeptides, which are important for the development of inflammation. Mice with genetic deletion of TRPV1 have been used to study the involvement of this receptor in the mediation of pain and inflammation in animal models of arthritis. However, the expression of TRPV1 in the mouse articular afferents has not been studied. We here provide numerical data on expression of TRPV1 in an identified population of sensory afferents to the mouse L3-L5 DRG that innervate joints, in comparison with that from bladder and skin. A combination of tracing and immunohistochemistry revealed that TRPV1-positive fibers innervate the mouse knee and ankle. At the level of DRG, approximately 40% of articular afferents from these joints express TRPV1 and the majority of them are peptidergic, as revealed by simultaneous immunostaining for the neuropeptide calcitonin gene-related peptide. These findings are consistent with the idea that activation of TRPV1 in peripheral axons of joint afferents may mediate the synovial release of neuropeptides in arthritis.

Smaller dendritic spines, weaker synaptic transmission, but enhanced spatial learning in mice lacking Shank1.

Experience-dependent changes in the structure of dendritic spines may contribute to learning and memory. Encoded by three genes, the Shank family of postsynaptic scaffold proteins are abundant and enriched in the postsynaptic density (PSD) of central excitatory synapses. When expressed in cultured hippocampal neurons, Shank promotes the maturation and enlargement of dendritic spines. Recently, Shank3 has been genetically implicated in human autism, suggesting an important role for Shank proteins in normal cognitive development. Here, we report the phenotype of Shank1 knock-out mice. Shank1 mutants showed altered PSD protein composition; reduced size of dendritic spines; smaller, thinner PSDs; and weaker basal synaptic transmission. Standard measures of synaptic plasticity were normal. Behaviorally, they had increased anxiety-related behavior and impaired contextual fear memory. Remarkably, Shank1-deficient mice displayed enhanced performance in a spatial learning task; however, their long-term memory retention in this task was impaired. These results affirm the importance of Shank1 for synapse structure and function in vivo, and they highlight a differential role for Shank1 in specific cognitive processes, a feature that may be relevant to human autism spectrum disorders.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat.

Trigeminal primary afferents expressing P2X(3) receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X(3)-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X(3) (+) terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN.

Presynaptic low- and high-affinity kainate receptors in nociceptive spinal afferents.

Presynaptic ionotropic glutamate receptors are increasingly attributed a role in the modulation of sensory input at the first synapse of dorsal root ganglion (DRG) neurons in the spinal dorsal horn. Central terminals of DRG neurons express AMPA and NMDA receptors whose activation modulates the release of glutamate, the main transmitter at these synapses. Previous work, with an antibody that recognizes all low-affinity kainate receptor subunits (GluR5, 6, 7), provided microscopic evidence of presynaptic kainate receptors in unidentified primary afferent terminals in superficial laminae of the spinal dorsal horn (Hwang SJ, Pagliardini S, Rustioni A, Valtschanoff JG. Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord. J Comp Neurol 2001; 436: pp. 275-289). We show here that, although all such subunits may be expressed in these terminals, GluR5 is the subunit most readily detectable at presynaptic sites in sections processed for immunocytochemistry. We also show that the high-affinity kainate receptor subunits KA1 and KA2 are expressed in central terminals of DRG neurons and are co-expressed with low-affinity receptor subunits in the same terminals. Quantitative data show that kainate-expressing DRG neurons are about six times more likely to express the P2X(3) subunit of the purinergic receptor than to express substance P. Thus, nociceptive afferents that express presynaptic kainate receptors are predominantly non-peptidergic, suggesting a role for these receptors in the modulation of neuropathic rather than inflammatory pain.

The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive.

The rat urinary bladder is innervated by neurons in dorsal root ganglia (DRG) that express the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), and a fraction of bladder afferents can bind the non-peptidergic marker isolectin B4 (IB4). We used histochemical binding and axonal tracing to identify the bladder afferents, and immunocytochemistry to determine the degree of colocalization of CGRP with IB4 in their cell bodies in DRG and in their central axons in the spinal cord. In the L6 DRG, about 60% of CGRP-positive neurons were also positive for IB4. In the spinal cord, IB4 and CGRP colocalized in fibers and terminals in the inner part of lamina II, the lateral collateral path, and the sacral parasympathetic nucleus (SPN). In SPN, the majority of IB4-positive fibers and terminals were also CGRP-positive. After injection of IB4 into the bladder wall, immunoreaction for IB4 was detected in SPN, but not in lamina II. These results suggest that most IB4-positive afferents from the bladder are also CGRP-positive, and that the distinction between peptidergic and non-peptidergic bladder afferents based on IB4 binding is of limited validity.

Expression of the vanilloid receptor TRPV1 in rat dorsal root ganglion neurons supports different roles of the receptor in visceral and cutaneous afferents.

A combination of tracing and multiple color immunofluorescence revealed that 69% of rat dorsal root ganglion (DRG) neurons innervating the urinary bladder expressed the vanilloid receptor TRPV1. In contrast, only 32% of DRG neurons innervating the skin of the L6 dermatome expressed TRPV1. However, a similar fraction of visceral (60-62%) and of cutaneous (59-60%) TRPV1-positive DRG neurons expressed the peptidergic markers substance P and calcitonin gene-related peptide, while the fraction of TRPV1-positive neurons that was labeled by the non-peptidergic marker Isolectin B4 was 58% for cutaneous and only 24% for visceral afferents. These results underscore differences of expression of functional markers in visceral and cutaneous afferents and support different mechanisms of activation of TRPV1 in viscera and in skin.

Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn.

Ionotropic glutamate receptors (IGR), including NMDA, AMPA, and kainate receptors, are expressed in terminals with varied morphology in the superficial laminae (I-III) of the dorsal horn of the spinal cord. Some of these terminals can be identified as endings of primary afferents, whereas others establish symmetric synapses, suggesting that they may be gamma-aminobutyric acid (GABA)-ergic. In the present study, we used confocal and electron microscopy of double immunostaining for GAD65, a marker for GABAergic terminals, and for subunits of IGRs to test directly whether IGRs are expressed in GABAergic terminals in laminae I-III of the dorsal horn. Although colocalization is hard to detect with confocal microscopy, electron microscopy reveals a substantial number of terminals immunoreactive for GAD65 also stained for IGRs. Among all GAD65-immunoreactive terminals counted, 37% express the NMDA receptor subunit NR1; 28% are immunopositive using an antibody for the GluR2/4 subunits of the AMPA receptor; and 20-35% are immunopositive using antibodies for the kainate receptor subunits GluR5, GluR6/7, KA1, or KA2. Terminals immunoreactive for IGR subunits and GAD65 establish symmetric synapses onto dendrites and perikarya and can be presynaptic to primary afferent terminals within both type 1 and type 2 synaptic glomeruli. Activation of presynaptic IGR may reduce neurotransmitter release. As autoreceptors in terminals of Adelta and C afferent fibers in laminae I-III, presynaptic IGRs may play a role in inhibiting nociception. As heteroreceptors in GABAergic terminals in the same laminae, on the other hand, presynaptic IGRs may have an opposite role and even contribute to central sensitization and hyperalgesia.

Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors.

The vanilloid receptor VR1 (TRPV1) is a temperature- and capsaicin-sensitive cation channel expressed by a class of primary afferents involved in nociception. To confirm the hypothesis that VR1-positive primary afferents are glutamatergic and contact spinal neurons that express the main classes of ionotropic glutamate receptors, we performed multiple immunofluorescent staining for VR1 and the glutamate transporter VGLUT2 (a specific marker for glutamatergic transmission) or AMPA and NMDA receptor subunits. VR1-positive cells in the dorsal root ganglion and boutons of their central afferent fibers in the dorsal horn expressed VGLUT2, and the latter contacted AMPA- or NMDA receptor-positive perikarya. Based on our previous observations of preferential targeting of VR1-positive primary afferents to spinal neurons that express the neurokinin receptor NK1 (Hwang et al., 2003), we further quantified the frequency of termination of VR1-positive afferents onto NK1-positive neurons co-expressing glutamate receptors. A larger fraction of NK1/NMDA receptors-positive than NK1/AMPA receptors-positive sites were contacted by VR1-positive boutons. We conclude that VR1-positive primary afferents in the rat use glutamate as neurotransmitter and contact postsynaptic sites that co-express NK1 and ionotropic glutamate receptors.

Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei.

Little is known about the central projection patterns of trigeminal afferent neurons expressing the vanilloid receptor TRPV1 and their coexpression of neuromodulatory peptides. To address these issues, we examined the distribution of TRPV1-positive neurons in the trigeminal ganglion (TG) and trigeminal sensory nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc) in the rat via light and electron microscopy. In addition, we studied the colocalization of TRPV1-positive neurons with substance P (SP) and calcitonin gene-related peptide (CGRP) via confocal microscopy. In TG, only small and medium-sized neurons were immunopositive for TRPV1. The staining for TRPV1 was found in axon collaterals in the dorsal parts of Vp, Vo, and Vi and in terminals and fibers throughout lamina I and the outer zone of lamina II (IIo) of Vc. With electron microscopy, TRPV1-positive fibers in the ascending and descending trigeminal tracts were found to be unmyelinated. Almost all TRPV1-positive terminals in Vc contained numerous large dense-core vesicles and formed synaptic contacts with single small dendrites. Multiple immunofluorescence revealed a high degree of colocalization of TRPV1 with SP and CGRP in TG neurons as well as in fibers and terminals confined to laminae I and IIo of Vc. These results suggest that the central projections of unmyelinated (C) afferents sensitive to noxious heat and capsaicin are organized differently between Vc and the rostral trigeminal nuclei and that Vc may play a role in the development of hyperalgesia.

A critical role for palladin in astrocyte morphology and response to injury.

Astrocytes respond to injury of the CNS with a dramatic change in morphology, contributing to the formation of a glial scar. We recently identified a novel actin-associated protein named palladin, which possesses the features of a potent cytoskeletal scaffold. Palladin expression was assayed in two populations of cultured astrocytes, polygonal versus stellate, and was detected at high levels in polygonal astrocytes and low levels in stellate astrocytes. When stellate astrocyte monolayers were wounded, palladin was rapidly upregulated along the edge of the wound, coordinate with an increase in actin assembly. Palladin upregulation occurred along a similar rapid time course following injury to the cerebral cortex of adult rats. To explore palladin function more directly, palladin cDNA was transfected into stellate astrocytes, which acquired a spread morphology and prominent actin bundles. These results suggest that palladin upregulation following injury may be a key step in the acquisition of the reactive astrocyte morphology.

Primary afferent terminals that express presynaptic NR1 in rats are mainly from myelinated, mechanosensitive fibers.

Presynaptic N-methyl-D-aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383-8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721-724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double-stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1-immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non-peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene-related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60-65% of all synapses in which it was expressed pre- or postsynaptically, or both, in spinal laminae I-IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception.

VR1-positive primary afferents contact NK1-positive spinoparabrachial neurons.

Neurons in rat superficial dorsal horn that express neurokinin receptor 1 (NK1), a receptor for substance P, play a critical role in the development of hyperalgesia. Thermal hyperalgesia is dramatically reduced after ablation of these neurons, but, paradoxically, not in mice that lack the NK1 receptor (Mantyh et al. [1997] Science 278:275-279). Because primary afferents that express vanilloid receptor 1 (VR1), a receptor for noxious heat, are essential for thermal nociception and hyperalgesia, we reasoned that VR1-positive fibers may terminate onto NK1-expressing dorsal horn neurons. We therefore combined immunofluorescent staining for VR1 and NK1 to show that NK1-positive neurons in lamina I are contacted by VR1-positive fibers. That these contacts represent synapses was verified by staining for the presynaptic marker synaptophysin and by electron microscopy. By combining retrograde tracing with immunocytochemistry, we also found that most NK1-positive cells contacted by VR1-positive fibers project to the lateral parabrachial nucleus. Because quantitative evaluation suggests a preferential targeting of NK1-positive lamina I neurons by fibers containing VR1, these results demonstrate a significant monosynaptic innervation of spinoparabrachial neurons by VR1-positive afferents.

Ligand-dependent recruitment of the ErbB4 signaling complex into neuronal lipid rafts.

Neuregulin (NRG) regulates synapse formation and synaptic plasticity, but little is known about the regulation of NRG signaling at synapses. Here we show that the NRG receptor ErbB4 was localized in anatomically defined postsynaptic densities in the brain. In cultured cortical neurons, ErbB4 was recruited to the neuronal lipid raft fraction after stimulation by NRG. Along with ErbB4, adaptor proteins Grb2 and Shc were translocated to lipid rafts by NRG stimulation. In transfected human embryonic kidney 293 cells, the partitioning of ErbB4 into a detergent-insoluble fraction that includes lipid rafts was increased by PSD-95 (postsynaptic density-95), through interaction of the ErbB4 C terminus with the PDZ [PSD-95/Discs large/zona occludens-1] domains of PSD-95. Disruption of lipid rafts inhibited NRG-induced activation of Erk and prevented NRG-induced blockade of induction of long-term potentiation at hippocampal CA1 synapses. Thus, our results indicate that NRG stimulation causes translocation of ErbB4 into lipid rafts and that lipid rafts are necessary for signaling by ErbB4.

Interaction between liprin-alpha and GIT1 is required for AMPA receptor targeting.

Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes.

Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha.

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons.