Pim van Nierop

Crystal Structure of Nicotinic Acetylcholine Receptor Homolog Achbp in Complex with an Alpha-Conotoxin Pnia Variant

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup α-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 Å of α-Ctx PnIA (A10L D14K), a potent blocker of the α7-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. α-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

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.

The Synaptic Proteome during Development and Plasticity of the Mouse Visual Cortex

During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood.

Identification and Functional Expression of a Family of Nicotinic Acetylcholine Receptor Subunits in the Central Nervous System of the Mollusc Lymnaea stagnalis

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the α-type, 1 is of the β-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The α-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise ∼10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

Profiling the human hippocampal proteome at all pathologic stages of Alzheimer's disease.

We performed a comprehensive quantitative proteomics study on human hippocampus tissue involving all Braak stages to assess changes in protein abundance over the various stages of Alzheimers disease (AD).

Reducing hippocampal extracellular matrix reverses early memory deficits in a mouse model of Alzheimer’s disease

Alzheimer’s disease is caused by increased production or reduced clearance of amyloid-β, which results in the formation amyloid-β plaques and triggers a cascade of downstream events leading to progressive neurodegeneration. The earliest clinical symptoms of Alzheimer’s disease, i.e., memory loss, are however poorly understood from a molecular and cellular perspective. Here we used APPswe/PS1dE9 (APP/PS1) transgenic mice to study the early pre-pathological effects of increased amyloid-β levels on hippocampal synaptic plasticity and memory. Using an unbiased proteomics approach we show that the early increase in amyloid-β levels in APP/PS1 mice at three months of age coincides with a robust and significant upregulation of several protein components of the extracellular matrix in hippocampal synaptosome preparations. This increase in extracellular matrix levels occurred well before the onset of plaque formation and was paralleled by impairments in hippocampal long-term potentiation and contextual memory. Direct injection into the hippocampus of the extracellular matrix inactivating enzyme chondroitinase ABC restored both long-term potentiation and contextual memory performance. These findings indicate an important role for the extracellular matrix in causing early memory loss in Alzheimer’s disease.

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.

SynSysNet: integration of experimental data on synaptic protein–protein interactions with drug-target relations

We created SynSysNet, available online at http://bioinformatics.charite.de/synsysnet, to provide a platform that creates a comprehensive 4D network of synaptic interactions. Neuronal synapses are fundamental structures linking nerve cells in the brain and they are responsible for neuronal communication and information processing. These processes are dynamically regulated by a network of proteins. New developments in interaction proteomics and yeast two-hybrid methods allow unbiased detection of interactors. The consolidation of data from different resources and methods is important to understand the relation to human behaviour and disease and to identify new therapeutic approaches. To this end, we established SynSysNet from a set of ∼1000 synapse specific proteins, their structures and small-molecule interactions. For two-thirds of these, 3D structures are provided (from Protein Data Bank and homology modelling). Drug-target interactions for 750 approved drugs and 50 000 compounds, as well as 5000 experimentally validated protein–protein interactions, are included. The resulting interaction network and user-selected parts can be viewed interactively and exported in XGMML. Approximately 200 involved pathways can be explored regarding drug-target interactions. Homology-modelled structures are downloadable in Protein Data Bank format, and drugs are available as MOL-files. Protein–protein interactions and drug-target interactions can be viewed as networks; corresponding PubMed IDs or sources are given.

Hippocampal Extracellular Matrix Levels and Stochasticity in Synaptic Protein Expression Increase with Age and Are Associated with Age-dependent Cognitive Decline

Age-related cognitive decline is a serious health concern in our aging society. Decreased cognitive function observed during healthy brain aging is most likely caused by changes in brain connectivity and synaptic dysfunction in particular brain regions. Here we show that aged C57BL/6J wild-type mice have hippocampus-dependent spatial memory impairments. To identify the molecular mechanisms that are relevant to these memory deficits, we investigated the temporal profile of mouse hippocampal synaptic proteome changes at 20, 40, 50, 60, 70, 80, 90, and 100 weeks of age. Extracellular matrix proteins were the only group of proteins that showed robust and progressive up-regulation over time. This was confirmed by immunoblotting and histochemical analysis, which indicated that the increased levels of hippocampal extracellular matrix might limit synaptic plasticity as a potential cause of age-related cognitive decline. In addition, we observed that stochasticity in synaptic protein expression increased with age, in particular for proteins that were previously linked with various neurodegenerative diseases, whereas low variance in expression was observed for proteins that play a basal role in neuronal function and synaptic neurotransmission. Together, our findings show that both specific changes and increased variance in synaptic protein expression are associated with aging and may underlie reduced synaptic plasticity and impaired cognitive performance in old age.

Time-dependent changes in the mouse hippocampal synaptic membrane proteome after contextual fear conditioning

A change in efficacy of hippocampal synapses is critical for memory formation. So far, the molecular analysis of synapses during learning has focused on small groups of proteins, whereas the dynamic global changes at these synapses have remained unknown. Here, we analyzed the temporal changes of the mouse hippocampal synaptic membrane proteome 1 and 4 h after contextual fear learning, comparing two groups; (1) a fear memory forming “delayed‐shock” group and (2) a fear memory‐deficient “immediate‐shock” group. No changes in protein expression were observed 1 h after conditioning between the two experimental groups. However, 423 proteins were significantly regulated 4 h later of which 164 proteins showed a temporal regulation after a delayed shock and 273 proteins after the stress of an immediate shock. From the proteins that were differentially regulated between the delayed‐ and the immediate‐shock groups at 4 h, 48 proteins, most prominently representing endocytosis, (amphiphysin, dynamin, and synaptojanin1), glutamate signaling (glutamate [NMDA] receptor subunit epsilon‐1, disks large homolog 3), and neurotransmitter metabolism (excitatory amino acid transporter 1, excitatory amino acid transporter 2, sodium‐ and chloride‐dependent GABA transporter 3) were regulated in both protocols, but in opposite directions, pointing toward an interaction of learning and stress. Taken together, this data set yields novel insight into diverse and dynamic changes that take place at hippocampal synapses over the time course of contextual fear‐memory learning.