Yvonne Gouwenberg

Local synthesis of actin-binding protein beta-thymosin regulates neurite outgrowth

Local protein synthesis plays an essential role in the regulation of various aspects of axonal and dendritic function in adult neurons. At present, however, there is no direct evidence that local protein translation is functionally contributing to neuronal outgrowth. Here, we identified the mRNA encoding the actin-binding protein β-thymosin as one of the most abundant transcripts in neurites of outgrowing neurons in culture. β-Thymosin mRNA is not evenly distributed in neurites, but appears to accumulate at distinct sites such as turning points and growth cones. Using double-stranded RNA knockdown, we show that reducing β-thymosin mRNA levels results in a significant increase in neurite outgrowth, both in neurites of intact cells and in isolated neurites. Together, our data demonstrate that local synthesis of β-thymosin is functionally involved in regulating neuronal outgrowth.

Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ∼1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, α-crystallin B, α-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.

Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs.

Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates.

Ventromedial Prefrontal Cortex Pyramidal Cells Have a Temporal Dynamic Role in Recall and Extinction of Cocaine-Associated Memory

In addicts, associative memories related to the rewarding effects of drugs of abuse can evoke powerful craving and drug seeking urges, but effective treatment to suppress these memories is not available. Detailed insight into the neural circuitry that mediates expression of drug-associated memory is therefore of crucial importance. Substantial evidence from rodent models of addictive behavior points to the involvement of the ventromedial prefrontal cortex (vmPFC) in conditioned drug seeking, but specific knowledge of the temporal role of vmPFC pyramidal cells is lacking. To this end, we used an optogenetics approach to probe the involvement of vmPFC pyramidal cells in expression of a recent and remote conditioned cocaine memory. In mice, we expressed Channelrhodopsin-2 (ChR2) or Halorhodopsin (eNpHR3.0) in pyramidal cells of the vmPFC and studied the effect of activation or inhibition of these cells during expression of a cocaine-contextual memory on days 1–2 (recent) and ∼3 weeks (remote) after conditioning. Whereas optical activation of pyramidal cells facilitated extinction of remote memory, without affecting recent memory, inhibition of pyramidal cells acutely impaired recall of recent cocaine memory, without affecting recall of remote memory. In addition, we found that silencing pyramidal cells blocked extinction learning at the remote memory time-point. We provide causal evidence of a critical time-dependent switch in the contribution of vmPFC pyramidal cells to recall and extinction of cocaine-associated memory, indicating that the circuitry that controls expression of cocaine memories reorganizes over time.

Neuregulin-3 in the mouse medial prefrontal cortex regulates impulsive action

BACKGROUND A deficit in impulse control is a prominent, heritable symptom in several psychiatric disorders, such as addiction, attention-deficit/hyperactivity disorder, and schizophrenia. Here, we aimed to identify genes regulating impulsivity, specifically of impulsive action, in mice. METHODS Using the widely used 5-choice serial reaction time task, we measured impulsive action in 1) a panel of 41 BXD recombinant inbred strains of mice (n = 13.7 ± .8 per strain; n = 654 total) to detect underlying genetic loci; 2) congenic mice (n = 23) to replicate the identified locus; 3) mice overexpressing the Nrg3 candidate gene in the medial prefrontal cortex (n = 21); and 4) a Nrg3 loss-of-function mutant (n = 59) to functionally implicate the Nrg3 candidate gene in impulsivity. RESULTS Genetic mapping of impulsive action in the BXD panel identified a locus on chromosome 14 (34.5-41.4 Mb), syntenic with the human 10q22-q23 schizophrenia-susceptibility locus. Congenic mice carrying the impulsivity locus (Impu1) confirmed its influence on impulsive action. Increased impulsivity was associated with increased Nrg3 gene expression in the medial prefrontal cortex (mPFC). Viral overexpression of Nrg3 in the mPFC increased impulsivity, whereas a constitutive Nrg3 loss-of-function mutation decreased it. CONCLUSIONS The causal relation between Nrg3 expression in the mPFC and level of impulsive action shown here provides a mechanism by which polymorphism in NRG3 in humans contributes to a specific cognitive deficit seen in several psychiatric diseases, such as addiction, attention-deficit/hyperactivity disorder, and schizophrenia.

Changes in molecular composition of rat medial prefrontal cortex synapses during adolescent development

Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)‐based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34–P78), with an over‐representation of cell‐membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44–P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non‐stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.

Postsynaptic expression of an epidermal growth factor receptor regulates cholinergic synapse formation between identified molluscan neurons

Epidermal growth factor (EGF) family members are conserved in both vertebrates and invertebrates. Recent studies suggest that EGF ligands in invertebrates may have neurotrophic actions that possibly compensate for the apparent absence of neurotrophins in these species. In this study, we have cloned an EGF receptor from the mollusk Lymnaea stagnalis (L‐EGFR), and shown that L‐EGFR is the receptor for a previously identified EGF‐like peptide in Lymnaea, named Lymnaea EGF (L‐EGF). Knock‐down of L‐EGFR expression prevented L‐EGF‐induced excitatory synapse formation between identified cholinergic neuron visceral dorsal 4 (VD4) and its postsynaptic partner left pedal dorsal 1 (LPeD1). Moreover, knock‐down of L‐EGFR also prevented synapse formation induced by Lymnaea brain conditioned medium, suggesting that L‐EGF is the most important, if not the only, brain‐derived factor that promotes excitatory cholinergic synapse formation in Lymnaea. Thus, our data establish canonical EGF/EGFR signaling as an important synaptotrophic mechanism in invertebrates.

The Extracellular Matrix Protein Brevican Limits Time-Dependent Enhancement of Cocaine Conditioned Place Preference

Cocaine-associated environmental cues sustain relapse vulnerability by reactivating long-lasting memories of cocaine reward. During periods of abstinence, responding to cocaine cues can time-dependently intensify a phenomenon referred to as ‘incubation of cocaine craving’. Here, we investigated the role of the extracellular matrix protein brevican in recent (1 day after training) and remote (3 weeks after training) expression of cocaine conditioned place preference (CPP). Wild-type and Brevican heterozygous knock-out mice, which express brevican at ~50% of wild-type levels, received three cocaine–context pairings using a relatively low dose of cocaine (5 mg/kg). In a drug-free CPP test, heterozygous mice showed enhanced preference for the cocaine-associated context at the remote time point compared with the recent time point. This progressive increase was not observed in wild-type mice and it did not generalize to contextual-fear memory. Virally mediated overexpression of brevican levels in the hippocampus, but not medial prefrontal cortex, of heterozygous mice prevented the progressive increase in cocaine CPP, but only when overexpression was induced before conditioning. Post-conditioning overexpression of brevican did not affect remote cocaine CPP, suggesting that brevican limited the increase in remote CPP by altering neuro-adaptive mechanisms during cocaine conditioning. We provide causal evidence that hippocampal brevican levels control time-dependent enhancement of cocaine CPP during abstinence, pointing to a novel substrate that regulates incubation of responding to cocaine-associated cues.

Strain Differences in Presynaptic Function PROTEOMICS, ULTRASTRUCTURE, AND PHYSIOLOGY OF HIPPOCAMPAL SYNAPSES IN DBA/2J AND C57Bl/6J MICE

Background: The inbred mouse strain DBA/2J shows impaired hippocampal memory formation, which has been attributed to postsynaptic changes. Results: DBA/2J shows reduced expression of exocytosis proteins, paired-pulse facilitation, and number of synaptic vesicles. Conclusion: Proteomic, ultrastructural, and physiological investigations suggest a deficit in presynaptic function in DBA/2J. Significance: This is the first study describing the DBA/2J hippocampal proteome and ultrastructure. The inbred strains C57BL/6J and DBA/2J (DBA) display striking differences in a number of behavioral tasks depending on hippocampal function, such as contextual memory. Historically, this has been explained through differences in postsynaptic protein expression underlying synaptic transmission and plasticity. We measured the synaptic hippocampal protein content (iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and mass spectrometry), CA1 synapse ultrastructural morphology, and synaptic functioning in adult C57BL/6J and DBA mice. DBA mice showed a prominent decrease in the Ras-GAP calcium-sensing protein RASAL1. Furthermore, expression of several presynaptic markers involved in exocytosis, such as syntaxin (Stx1b), Ras-related proteins (Rab3a/c), and rabphilin (Rph3a), was reduced. Ultrastructural analysis of CA1 hippocampal synapses showed a significantly lower number of synaptic vesicles and presynaptic cluster size in DBA mice, without changes in postsynaptic density or active zone. In line with this compromised presynaptic morphological and molecular phenotype in DBA mice, we found significantly lower paired-pulse facilitation and enhanced short term depression of glutamatergic synapses, indicating a difference in transmitter release and/or refilling mechanisms. Taken together, our data suggest that in addition to strain-specific postsynaptic differences, the change in dynamic properties of presynaptic transmitter release may underlie compromised synaptic processing related to cognitive functioning in DBA mice.

Impact of genetic variation on synaptic protein levels in genetically diverse mice.

The relative abundance of synaptic proteins shapes protein complex formation and is essential for synapse function and behavioral fitness. Here, we have used a panel of highly diverse inbred strains of mice—NOD/LtJ, A/J, 129S1/SvImJ, FVB/NJ, C57BL/6J, WSB/EiJ, PWK/PhJ, and CAST/EiJ—to quantify the effects of genetic variation on the synaptic proteome between strains. Using iTRAQ‐based quantitative proteome analyses, we detected significant differences in ∼20% of 400 core synaptic proteins. Surprisingly, the differentially abundant proteins showed a modest range of variation across strains, averaging about 1.3‐fold. Analysis of protein abundance covariation across the eight strains identified known protein–protein relations (proteins of Arp2/3 complex), as well as novel relations (e.g. Dlg family, Fscn1). Moreover, covariation of synaptic proteins was substantially tighter (∼fourfold more dense than chance level) than corresponding networks of synaptic transcripts (∼twofold more dense than chance). The tight stoichiometry and coherent synaptic protein covariation networks suggest more intense evolutionary selection at this level of molecular organization. In conclusion, genetic diversity in the mouse genome differentially affects the transcriptome and proteome, and only partially penetrates the synaptic proteome. Protein abundance correlation analyses in genetically divergent strains can complement protein–protein interaction network analyses, to provide insight into protein interactomes.