Targeting Of Synaptic Proteins Research Articles

CTTNBP2 Controls Synaptic Expression of Zinc-Related Autism-Associated Proteins and Regulates Synapse Formation and Autism-like Behaviors

Synaptic dysregulation is a critical feature of autism spectrum disorders (ASDs). Among various autism-associated genes, cortactin binding protein 2 (CTTNBP2) is a cytoskeleton regulator predominantly expressed in neurons and highly enriched at dendritic spines. Here, using Cttnbp2 knockout and ASD-linked mutant mice, we demonstrate that Cttnbp2 deficiency reduces zinc levels in the brain, alters synaptic protein targeting, impairs dendritic spine formation and ultrastructure of postsynaptic density, and influences neuronal activation and autism-like behaviors. A link to autism, the NMDAR-SHANK pathway, and zinc-related regulation are three features shared by CTTNBP2-regulated synaptic proteins. Zinc supplementation rescues the synaptic expression of CTTNBP2-regulated proteins. Moreover, zinc supplementation and administration of D-cycloserine, an NMDAR coagonist, improve the social behaviors of Cttnbp2-deficient mice. We suggest that CTTNBP2 controls the synaptic expression of a set of zinc-regulated autism-associated genes and influences NMDAR function and signaling, providing an example of how genetic and environmental factor crosstalk controls social behaviors.

‐The Function of CASK in Transcriptional Regulation in Neurons

CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.Support or Funding InformationThis project has been supported by CABRI Undergraduate Research Fellowship

Neurobeachin, a Regulator of Synaptic Protein Targeting, Is Associated with Body Fat Mass and Feeding Behavior in Mice and Body-Mass Index in Humans

Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/− mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.

Multiple roles of the Rho GEF ephexin1 in synapse remodeling

Synapse remodeling, which involves changes in the synaptic structure and their molecular composition, is required for the maturation and refinement of neural circuits. Although synapse remodeling is known to be tightly dependent on the assembly of local actin cytoskeleton, how actin directs the structural changes of synapse and targeting of synaptic proteins are not fully understood. Recently, we identified ephexin1, a Rho guanine nucleotide exchange factor (GEF) that regulates actin dynamics, plays an essential role in the maturation and functioning of the mammalian neuromuscular junction (NMJ). We showed that ephexin1 regulates the synaptic organization of the neurotransmitter receptor acetylcholine receptor (AChR) clusters through RhoA-dependent actin reorganization. Interestingly, ephexin1 has been implicated in the regulation of postsynaptic structure as well as the presynaptic vesicle release at various types of synapses. Our findings thus establish a novel function of ephexin1 in synapse remodeling through regulating the synaptic targeting of neurotransmitter receptors, revealing a versatile role of ephexin1 at synapses.

Synaptic protein targeting in presenilin animal models

Synaptic protein targeting in presenilin animal models

The role of lipid binding for the targeting of synaptic proteins into synaptic vesicles

Synaptic vesicles (SVs) are key structures for synaptic transmission in neurons. Numerous membrane-associated proteins are sorted from the Golgi complex to the axon and the presynaptic terminal. Protein-protein and protein-lipid interactions are involved with SV targeting in neurons. Interestingly, many SV proteins have lipid binding capability, primarily with either cholesterol or phosphoinositides (PIs). As examples, the major SV protein synaptophysin can bind to cholesterol, a major lipid component in SVs, while several other SV proteins, including synaptotagmin, can bind to PIs. Thus, lipid-protein binding plays a key role for the SV targeting of synaptic proteins. In addition, numerous SV proteins can be palmitoylated. Palmitoylation is thought to be another synaptic targeting signal. Here, we briefly describe the relationship between lipid binding and SV targeting. [BMB reports 2009; 42(1): 1-5].

CASK associates with glutamate receptor interacting protein and signaling molecules

CASK has been implicated in synaptic protein targeting, synaptic organization, and transcriptional regulation. Here, three more CASK associated proteins, GRIP1, PKCε, and RGS4, were initially identified by immunoprecipitation and mass analysis, and confirmed by immunoprecipitation–immunoblotting assay using rat brain extract. Via the interaction with GRIP1, GluR2/3 was also co-immunoprecipitated by CASK antibody from rat brain. The PDZ and SH3–GK domains of CASK were demonstrated as the associated domains for GRIP1 and PKCε, respectively. The associations between CASK, PKCε, and RGS4 were up-regulated in the adult brain compared with postnatal day 11 rat brain. In contrast, the associations of CASK with Mint1, GRIP1, and GluR2/3 were down-regulated in the adult brain. These results suggest that CASK protein complex is developmentally regulated by unknown signals. In conclusion, our study suggests that the CASK protein complex not only functions as a scaffold but also recruits signaling molecules and may contribute to signal transduction.

The Role of the MAGUK Protein CASK in Neural Development and Synaptic Function

CASK, which belongs to the family of membrane-associated guanylate kinase (MAGUK) proteins, is recognized as a multidomain scaffolding protein highly expressed in the mammalian nervous system. MAGUK proteins generally target to neuronal synapses and regulate trafficking, targeting, and signaling of ion channels. However, CASK is a unique MAGUK protein in several respects. It not only plays a role in synaptic protein targeting but also contributes to neural development and regulation of gene expression. Several CASK-interacting proteins have been identified from yeast two-hybrid screening and biochemical isolation. These proteins, whose interactions with CASK are reviewed here, include the Parkinson's disease molecule parkin, the adhesion molecule neurexin, syndecans, calcium channel proteins, the cytoplasmic adaptor protein Mint1, Veli/mLIN-7/MALS, SAP97, caskin and CIP98, transcription factor Tbr-1, and nucleosome assembly protein CINAP. More important, CASK may form different complexes with different binding partners and perform different functions. Among these interactions, CASK, Tbr-1, and CINAP can form a transcriptional complex regulating gene expression. Reelin and NMDAR subunit 2b (NR2b) genes have been identified as Tbr-1 target genes. Reelin is critical for neural development. NR2b is an important subunit of NMDAR, which plays important roles in neural function and neurological diseases. Regulation of reelin and NR2b expression suggests the potential roles of the Tbr-1-CASK-CINAP complex in neural activity, development, and disease. The functions of these CASK protein complexes are also discussed in detail in this review.

The CASK/Lin-2 Drosophila Homologue, Camguk, Could Play a Role in Epithelial Patterning and in Neuronal Targeting

Drosophila Camguk (Cmg) is a member of the CAMGUK subfamily of the MAGUK family of proteins which are localized at cell junction and other plasma membrane specialized regions, from worms to mammals. The protein structure of Cmg, as the other CAMGUK proteins, is characterized by only one PDZ domain and an additional CaM kinase domain, similar to CaMKII. While the mammalian ortholog CASKs play an important role in synaptic protein targeting and in synaptic plasticity, the Drosophila Cmg role is unknown. To study its potential role, we reported a detailed analysis of mRNA distribution of the Drosophila cmg gene at cellular and developmental level, during embryonic, larval, pupal and adult stages. The transient cmg transcription in midgut and Malpighian tubules may suggest a potential function in cell junction formation and in epithelial tissue patterning. Interestingly, cmg transcription increases substantially during embryonic neuroblast proliferation, becoming predominant in the developing central nervous system (CNS) during embryonic and postembryonic development stages and in the mature brain. In addition, a high transcriptional level was detected in the eye imaginal discs and in the adult retina, demonstrating a specific and continuous expression of cmg in neuroblasts and photoreceptor neurons, from the onset of cytodifferentiation. Our findings suggest that Cmg could play a potential role in transmenbrane protein targeting, particularly in synapses. These observations suggest the existence of a common highly conserved mechanism involved in forming and maintaining proper synaptic protein targeting, which are fundamental features of synaptic plasticity, learning and memory. Through its function, the CaM kinase domain-containing Cmg may be involved in signal transduction cascade. Its potential relation to Calmodulin and CaMKII is discussed.

The Golgi apparatus is present in perisynaptic, subependymal and perivascular processes of astrocytes and in processes of retinal Müller glia

The Golgi apparatus (GA) of innervated rat and chicken skeletal muscle is present in a typical perinuclear location, and in subsynaptic areas where it disperses after denervation. It was suggested that the subsynaptic segments of the GA are linked with functions involved in the maturation and targeting of synaptic proteins. Similarly, the GA of rat myocardium is found in a perinuclear location and between myofibrils, adjacent to the T system of tubules. These findings raise the question whether the GA of polarized cells is present in a typical perinuclear location, for the performance of general “housekeeping” functions, and in distal areas, for the mediation of specialized functions. Astrocytes may contain GA within their long cytoplasmic processes which are difficult to identify in thin sections. To ensure the astrocytic origin of GA in otherwise unidentifiable small processes, we used transgenic mice expressing the rat MG160 medial Golgi sialoglycoprotein only in the GA of astrocytes, and visualized the GA with monoclonal antibody 10A8 (mAb10A8) which reacts only with rat MG160. Thus, we identified cisternae of the GA in distal perisynaptic and subependymal processes, in perivascular foot plates of cerebral astrocytes, and in processes of the Müller glia in the retina. A similar strategy may be adopted in future investigations aiming at the detection of elements of the GA in distal processes of neurons and oligodendrocytes. The functional implications of GA in perisynaptic astrocytic processes and other processes are unknown. However, the isolation and molecular characterization of the perisynaptic subset of astrocytic Golgi may be feasible, since others have purified the astrocytic glutamate transporter 1 (GLT1) from crude synaptosomal fractions in which astrocytic processes are probably unavoidable contaminants.

The low molecular weight guanosine triphosphate-binding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes

The Rab genes have recently been cloned and sequenced in mammals, and their products represent good candidates for low molecular weight guanosine triphosphate-binding proteins involved in the regulation of intracellular transport of vesicles in higher eukaryotes. Remarkably, each of the Rab proteins appears to be associated with a distinct step of either the exocytic or endocytic pathway. In particular, Rab6p has been localized to the outermost Golgi cisternae in normal rat kidney cells, where its function remains unclear. In this work, we have carried out a series of immunocytochemical analyses of the subcellular distribution of Rab6p in a polarized cell, the electrocyte of Torpedo marmorata, to elucidate the molecular mechanisms involved in the sorting and targeting of synaptic proteins. We report that, aside from its Golgi localization, the bulk of Rab6p associates with clusters of post-Golgi vesicles, primarily those located at the cytoplasmic face of the innervated membrane of the electrocyte. Consequently, Rab6p presents a polarized distribution in this cell. Furthermore, we show that this distribution is dependent upon the integrity of the microtubule network of the electrocyte. These data are coherent with the notion that Rab6p is involved in the regulation of membrane traffic from the trans-Golgi network to the innervated plasma membrane, delivering, by way of a microtubule-based organelle transport mechanism, synaptic proteins to their appropriate locations.