Naweed I. Syed

A glia-derived acetylcholine-binding protein that modulates synaptic transmission

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Transplantation and functional integration of an identified respiratory interneuron in lymnaea stagnalis

The possibility that damaged neural circuitries can be repaired through grafting has raised questions regarding the cellular mechanisms required for functional integration of transplanted neurons. Invertebrate models offer the potential to examine such mechanisms at the resolution of single identified neurons within well-characterized neural networks. Here it is reported that a specific deficit in the respiratory behavior of a pulmonate mollusc, caused by the ablation of a solitary interneuron, can be restored by grafting an identical donor interneuron. The transplanted interneuron not only survives and extends neurites within the host nervous system, but under specific conditions forms synapses with appropriate target neurons and is physiologically integrated into the hosts circuitry, thereby restoring normal behavior.

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.

CRNF, a Molluscan Neurotrophic Factor That Interacts with the p75 Neurotrophin Receptor

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

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.

Differential Proteomics Reveals Multiple Components in Retrogradely Transported Axoplasm After Nerve Injury

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of ∼4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.

Synaptogenesis in the CNS: An Odyssey from Wiring Together to Firing Together

To acquire a better comprehension of nervous system function, it is imperative to understand how synapses are assembled during development and subsequently altered throughout life. Despite recent advances in the fields of neurodevelopment and synaptic plasticity, relatively little is known about the mechanisms that guide synapse formation in the central nervous system (CNS). Although many structural components of the synaptic machinery are pre‐assembled prior to the arrival of growth cones at the site of their potential targets, innumerable changes, central to the proper wiring of the brain, must subsequently take place through contact‐mediated cell‐cell communications. Identification of such signalling molecules and a characterization of various events underlying synaptogenesis are pivotal to our understanding of how a brain cell completes its odyssey from ‘wiring together to firing together’. Here we attempt to provide a comprehensive overview that pertains directly to the cellular and molecular mechanisms of selection, formation and refinement of synapses during the development of the CNS in both vertebrates and invertebrates.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis

BackgroundThe freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.ResultsIn this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes.ConclusionThis study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

Mercury-induced toxicity of rat cortical neurons is mediated through N-methyl-D-Aspartate receptors

BackgroundMercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer’s disease, Parkinson’s disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid) on cultured, rat cortical neurons.ResultsWe found that inorganic mercuric chloride (HgCl2 –at 0.025 to 25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Whole-cell patch-clamp recordings of pyramidal neurons revealed that HgCl2 not only enhanced the amplitude and frequency of synaptic, inward currents, but also increased spontaneous synaptic potentials followed by sustained membrane depolarization. HgCl2 also triggered sustained, 2–5 fold rises in intracellular calcium concentration ([Ca2+]i). The observed increases in neuronal activity and [Ca2+]i were substantially reduced by the application of MK 801, a non-competitive antagonist of N-Methyl-D-Aspartate (NMDA) receptors. Importantly, our study further shows that a pre incubation or co-application of MK 801 prevents HgCl2-induced reduction of cell viability and a disruption of β-tubulin.ConclusionsCollectively, our data show that HgCl2-induced toxic effects on central neurons are triggered by an over-activation of NMDA receptors, leading to cytoskeleton instability.

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.