A synaptic molecular dependency network in knockdown of autism- and schizophrenia-associated genes revealed by multiplexed imaging

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Structural and functional studies on different human FABP types

Interaction of various ligands with recombinant proteins of 5 human FABP types was studied by radiochemical and fluorescence procedures. Liver, heart, intestinal and myelin FABP showed a higher affinity for oleic acid than adipocyte FABP. Intestinal and adipocyte FABP had a relatively high Kd value for arachidonic acid. Liver and intestinal FABP showed high affinity for DAUDA in contrast to the other FABP types. ANS was only well bound by liver and adipocyte FABP. Retinol was not bound by any FABP type, retinoic acid only by adipocyte FABP. Data indicate the importance of both electrostatic and hydrophobic interaction for the ligand-FABP binding. The immunological crossreactivity between six human FABP types including epidermal FABP and their respective antibodies raised in rabbit, chicken and mouse appeared to be low and may suggest heterogeneity of protein surface.     

Building a synapse

L-glutamate is the primary neurotransmitter at excitatory synapses in the vertebrate CNS and at arthropod neuromuscular junctions (NMJs). However, the molecular mechanisms that trigger the recruitment of glutamate receptors at the onset of synaptogenesis and promote their stabilization at postsynaptic densities remain poorly understood. We have reported the discovery of a novel, evolutionary conserved molecule, Neto, essential for clustering of ionotropic glutamate receptors (iGluRs) at Drosophila NMJ. Neto is the first auxiliary subunit described in Drosophila and is the only non-channel subunit absolutely required for functional iGluRs. Here we review the role of Drosophila Neto in synapse assembly, its similarities with other Neto proteins and a new perspective on how glutamatergic synapses are physically assembled and stabilized.     

Leukemia inhibitory factor influences the timing of programmed synapse withdrawal from neonatal muscles

We show that leukemia inhibitory factor (LIF) plays a physiological role in the programmed withdrawal of synapses form neonatal muscles. First, LIF mRNA is present in embryonic skeletal muscle and is developmentally regulated. We detect high levels of LIF mRNA at embryonic day 17 (E17) in mouse hind leg muscles. The content of LIF mRNA falls 10-fold between E17 and birth and then remains low in the neonate and adult. The decrease in LIF mRNA in skeletal muscle coincides with the end of secondary myogenesis and the completion of the adult number of myofibers. Second, treatment of the mouse tensor fascia latae (TFL), a superficial muscle of the hind leg, with LIF from birth (100 ng/day), transiently delays the withdrawal of excess inputs from polyneuronally innervated myofibers by approximately 3 days. The midpoint of the process is shifted from 7.5 ± 0.5 to 10.2 ± 0.6 days of age. LIF treatment delays synapse withdrawal by altering its timing without an appreciable effect on its rate. Third, in mice homozygous for a distruption of the LIF gene, the midpoint in the reduction of multiply innervated TFL myofibers occurs 1 day earlier, at 6.5 ± 0.5 days of age. Muscle fiber number is unchanged in LIF null mice. Treatment with LIF does not alter the rate of neonatal growth, the number of muscle fibers in the TFL, or the reappearance of inputs that have been eliminated. Instead, LIF appears to delay maturation of the motor unit by transiently delaying the onset of synapse withdrawal. We hypothesize that this is a necessary component of a selective process that will operate simultaneously and equally on multiple, competing motor units. © 1995 John Wiley & Sons, Inc.     

IDENTIFYING COEVOLUTIONARY PATTERNS IN HUMAN LEUKOCYTE ANTIGEN (HLA) MOLECULES

The antigenic peptide, major histocompatibility complex molecule (MHC; also called human leukocyte antigen, HLA), coreceptor CD8, or CD4 and T‐cell receptor (TCR) function as a complex to initiate effectors’ mechanisms of the immune system. The tight functional and physical interaction among these molecules may have involved strong coevolution links among domains within and between proteins. Despite the importance of unraveling such dependencies to understand the arms race of host–pathogen interaction, no previous studies have aimed at achieving such an objective. Here, we perform an exhaustive coevolution analysis and show that indeed such dependencies are strongly shaping the evolution and probably the function of these molecules. We identify intramolecular coevolution in HLA class I and II at domains important for their immune activity. Most of the amino acid sites identified to be coevolving in HLAI have been also detected to undergo positive Darwinian selection highlighting therefore their adaptive value. We also identify coevolution among antigen‐binding pockets (P1‐P9) and among these and TCR‐binding sites. Conversely to HLAI, coevolution is weaker in HLAII. Our results support that such coevolutionary patterns are due to selective pressures of host–pathogen coevolution and cooperative binding of TCRs, antigenic peptides, and CD8/CD4 to HLAI and HLAII.     

Calcineurin Aγ is a Functional Phosphatase That Modulates Synaptic Vesicle Endocytosis

Variation in PPP3CC, the gene that encodes the γ isoform of the calcineurin catalytic subunit, has been reported to be associated with schizophrenia. Because of its low expression level in most tissues, there has been little research devoted to the specific function of the calcineurin Aγ (CNAγ) versus the calcineurin Aα (CNAα) and calcineurin Aβ (CNAβ) catalytic isoforms. Consequently, we have a limited understanding of the role of altered CNAγ function in psychiatric disease. In this study, we demonstrate that CNAγ is present in the rodent and human brain and dephosphorylates a presynaptic substrate of calcineurin. Through a combination of immunocytochemistry and immuno-EM, we further show that CNAγ is localized to presynaptic terminals in hippocampal neurons. Critically, we demonstrate that RNAi-mediated knockdown of CNAγ leads to a disruption of synaptic vesicle cycling in cultured rat hippocampal neurons. These data indicate that CNAγ regulates a critical aspect of synaptic vesicle cycling and suggest that variation in PPP3CC may contribute to psychiatric disease by altering presynaptic function.     

Dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2

Mutations in the gene encoding Ca_v1.4, CACNA1F_, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Ca_v1.4 channels, there are defects in the development of “ribbon” synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Ca_v1.4 channels. Whether defects in PR synapse development due to altered Ca_v1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Ca_v1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Ca_v1.4-selective antibodies, we found that Ca_v1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Ca_v1.4 activation is either enhanced (Ca_v1.4_I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Ca_v1.4_I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Ca_v1.4 function for efficient PR synapse maturation, and that dysregulation of Ca_v1.4 channels in CSNB2 may have synaptopathic consequences.     

The Regulation of Synaptic Protein Turnover

Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and are modulated by neuronal activity in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but the understanding of protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, as well as the turnover rates of specific neuronal proteins.     

Regeneration of central and peripheral synaptic connections in the locomotor system of the pteropod molluscClione limacina

The neural network underlying rhythmic wing movements in the molluscClione limacina is well-studied. Two different groups of motoneurons innervate two distinct groups of wing muscles. The locomotor rhythm generated in the left and right pedal ganglia is synchronized by interneurons. When the axons of the locomotor motoneurons are crushed, numerous fine neurites sprout towards the denervated muscles and reach them in 8–15 days. At this stage motoneurons project to and synapse on not only correct but equally incorrect muscle targets. After 2 weeks of regeneration the number of incorrect neurites and synaptic connections begins to decrease and following 1.5–2 months all incorrect connections are eliminated, incorrect axons are withdrawn and the behavioral deficit is compensated. In this study the regeneration of interneurons and the growth profiles of inter- and motoneurons were also studiedin vitro. Two individually isolated pedal ganglia were co-cultured in three different configurations: a) the wing nerve stump from one ganglion was fixed against the commissural stump from another ganglion; b) the wing nerve stumps were fixed against each other; c) the commissural stumps were fixed against each other. Under the above experimental conditions we found that the interneurons were able to cross only the contact between two commissural stumps, and in this case found their original targets, restored correct connections and synchronized the rhythm in two pedal ganglia. In contrast, motoneurons were able to cross all types of contacts.