Cameron B. Gundersen

Presynaptic Dysfunction in Drosophila csp Mutants

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Suppression Cloning of the cDNA for a Candidate Subunit of a Presynaptic Calcium Channel

A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.

The effects of botulinum toxin on the synthesis, storage and release of acetylcholine

Abstract Botulinum toxin is a presynaptically-acting polypeptide neurotoxin. The toxin causes an almost complete inhibition of evoked transmitter release from cholinergic nerve endings. Characteristics inhibition by the toxin of the quantal and nonquantal components of resting ACh output have also been described. In this context botulinum toxin has been a valuable probe of the transmitter release process. Under certain circumstances an effect of the toxin on ACh synthesis has also been found. A postulate has been advanced which could account for these multiple actions of botulinum toxin, and several tests of this nodel have been outlined. Regardless of the validity of the proposal, the avenues of pursuit which it suggests could lead to a firmer understanding of the mechanism of action of botulinum toxin and of the functioning of cholinergic nerve terminals.

Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor Neurons

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

The nucleotide and deduced amino acid sequence of a rat cysteine string protein

Cysteine string proteins are novel, heavily lipidated components of synaptic vesicles. They have previously been studied in Drosophila (insect) and Torpedo (fish). To facilitate further investigation of the structure and function of these proteins in mammals, we isolated and sequenced the cDNA and conducted an initial characterization of a rat cysteine string protein. Nucleotide sequencing reveals that this rat protein is highly homologous to the insect and fish cysteine string proteins. At the amino acid level, the fish and rat proteins are 82% identical. The rat cysteine string protein is encoded by an approximately 5 kb mRNA that is ubiquitously expressed in rat brain. Using antibodies that cross-react with the rat protein, we find that the rat cysteine string protein is predominantly associated with nerve endings and synaptic vesicles. Moreover, like its Torpedo (fish) counterpart, it is extensively fatty acylated. It will be of considerable interest to ascertain the functional correlates of these cross-species similarities of cysteine string proteins.

Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

Abstract: Using sequential incubations in media of different K+ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic‐mass spectrometric (GC‐MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K+ and with 2 μM‐[2H9]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [2H4]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K+ solution on the metabolism during a second low or high K+ incubation of a [2H9]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [2H9]Ch metabolism versus [2H4]Ch metabolism. The most striking result we obtained was that [2H9]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K+ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K+ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K+ media. When labeled choline was present during the release incubation, a‘newly‐synthesized’pool of acetylcholine was formed in the tissue. In toxin‐treated slices exposed to high K+, both the production and the release of this‘newly‐synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [3H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxins interaction with the mechanisms regulating acetylcholine turnover.

Nerve Growth Factor Signals via Preexisting TrkA Receptor Oligomers

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.

Widespread expression of human cysteine string proteins

We present the nucleotide and deduced amino acid sequences of a human cysteine string protein (csp) and a unique truncated csp variant that derives from the retention of an exonic sequence that introduces a premature, in‐frame stop codon. Low stringency Southern analysis is compatible with the presence of a single human csp gene. Northern analysis reveals that human csp mRNA has both a more heterogeneous size distribution and a more widespread tissue distribution than previously reported for other caps. Exemplifying this is the fact that csp immunoreactivity is detected in Triton X‐114 extracts of human blood, an observation which may facilitate blood‐based diagnostic assays of csp status in man.

Lithium Ions Enhance Cysteine String Protein Gene Expression In Vivo and In Vitro

Abstract: Lithium is a well established pharmacotherapy for the treatment of recurrent manic‐depressive illness. However, the mechanism by which lithium exerts its therapeutic action remains elusive. Here we report that lithium at 1 mM significantly increased the expression of cysteine string proteins (CSPs) in a pheochromocytoma cell line (PC12 cells) differentiated by nerve growth factor. These cells concomitantly exhibited increased expression of CSPs in their cell bodies and boutons. Enhanced CSP expression was also observed in the brain of rats fed a lithium‐containing diet, which elevated serum lithium to a therapeutically relevant concentration of ∼1.0 mM. However, both in vitro and in vivo, the expression of another synaptic vesicle protein, synaptophysin, and the t‐SNARE, synaptosomal‐associated protein of 25 kDa (SNAP‐25), was not significantly altered by lithium. These observations indicate that lithium‐induced changes of CSP gene expression may contribute to the therapeutic efficacy of this monovalent cation.