Luis Polo-Parada

Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions

Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.

Distinct Roles of Different Neural Cell Adhesion Molecule (NCAM) Isoforms in Synaptic Maturation Revealed by Analysis of NCAM 180 kDa Isoform-Deficient Mice

Mice that lack all three major isoforms of neural cell adhesion molecule (NCAM) (180 and 140 kDa transmembrane, and 120 kDa glycosylphosphatidylinositol linked) were previously shown to exhibit major alterations in the maturation of their neuromuscular junctions (NMJs). Specifically, even by postnatal day 30, they failed to downregulate from along their axons and terminals an immature, brefeldin A-sensitive, synaptic vesicle-cycling mechanism that used L-type Ca2+ channels. In addition, these NCAM null NMJs were unable to maintain effective transmitter output with high-frequency repetitive stimulation, exhibiting both severe initial depression and subsequent cyclical periods of total transmission failures that were of presynaptic origin. As reported here, mice that lack only the 180 kDa isoform of NCAM downregulated the immature vesicle-cycling mechanism on schedule, implicating either the 140 or 120 kDa NCAM isoforms in this important maturational event. However, 180 NCAM-deficient mice still exhibited many functional transmission defects. Although 180 NCAM null NMJs did not show the severe initial depression of NCAM null NMJs, they still had cyclical periods of complete transmission failure. In addition, several presynaptic molecules were expressed at lower levels or were more diffusely localized. Thus, the 180 kDa isoform of NCAM appears to play an important role in the molecular organization of the presynaptic terminal and in ensuring effective transmitter output with repetitive stimulation. Our results also suggest that PKC and MLCK (myosin light chain kinase) may be downstream effectors of NCAM in these processes. Together, these results indicate that different isoforms of NCAM mediate distinct and important events in presynaptic maturation.

NCAM 180 acting via a conserved C-terminal domain and MLCK is essential for effective transmission with repetitive stimulation.

NCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions. The effects of KENESKA and other peptides on MLCK and MLC activation and on failures in both wild-type and NCAM 180 null junctions supported this pathway, and serine phosphorylation of KENESKA was critical. We propose that this pathway is required to replenish synaptic vesicles utilized during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones or their subsequent exocytosis.

Pee-dots: biocompatible fluorescent carbon dots derived from the upcycling of urine

We have demonstrated an easy, economic, one-step synthetic route to water-soluble fluorescent carbon dots derived from the thermal upcycling of urine. These “pee-dots” (PDs), which primarily comprise hydrophile-decorated amorphous carbon, exhibit bright, stable, excitation wavelength dependent fluorescence in aqueous solution and are shown to be useful nanoscale labels in cell imaging applications. Cytotoxicity studies demonstrate that these PDs are benign toward model cell lines, even at concentrations as high as 500 μg mL−1. Notably, this approach converts an otherwise useless, negatively-valued byproduct of human life into a value-added nanoscale product while simultaneously pasteurizing the waste stream. The reported PDs proved to be effective nanoprobes for the fluorescence-based detection of heavy metal ions of environmental concern, particularly Cu2+ and Hg2+ ions which were found to be strong quenchers of their fluorescence. Interestingly, the optical properties and nanoscale dimensions of the PDs are a direct reflection of the diet (e.g., vitamin C or asparagus (sulfur) fortified) followed by the urine donor.

Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives

BACKGROUND AND PURPOSE Electrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca2+ signalling.

Characterization of rhythmic Ca2+ transients in early embryonic chick motoneurons: Ca2+ sources and effects of altered activation of transmitter receptors

In the nervous system, spontaneous Ca2+ transients play important roles in many developmental processes. We previously found that altering the frequency of electrically recorded rhythmic spontaneous bursting episodes in embryonic chick spinal cords differentially perturbed the two main pathfinding decisions made by motoneurons, dorsal–ventral and pool-specific, depending on the sign of the frequency alteration. Here, we characterized the Ca2+ transients associated with these bursts and showed that at early stages while motoneurons are still migrating and extending axons to the base of the limb bud, they display spontaneous, highly rhythmic, and synchronized Ca2+ transients. Some precursor cells in the ependymal layer displayed similar transients. T-type Ca2+ channels and a persistent Na+ current were essential to initiate spontaneous bursts and associated transients. However, subsequent propagation of activity throughout the cord resulted from network-driven chemical transmission mediated presynaptically by Ca2+ entry through N-type Ca2+ channels and postsynaptically by acetylcholine acting on nicotinic receptors. The increased [Ca2+]i during transients depended primarily on L-type and T-type channels with a modest contribution from TRP (transient receptor potential) channels and ryanodine-sensitive internal stores. Significantly, the drugs used previously to produce pathfinding errors altered transient frequency but not duration or amplitude. These observations imply that different transient frequencies may differentially modulate motoneuron pathfinding. However, the duration of the Ca2+ transients differed significantly between pools, potentially enabling additional distinct pool-specific downstream signaling. Many early events in spinal motor circuit formation are thus potentially sensitive to the rhythmic Ca2+ transients we have characterized and to any drugs that perturb them.

Selective Targeting of Different Neural Cell Adhesion Molecule Isoforms during Motoneuron–Myotube Synapse Formation in Culture and the Switch from an Immature to Mature Form of Synaptic Vesicle Cycling

Characterization of neuromuscular junction formation and function in mice lacking all neural cell adhesion molecule (NCAM) isoforms or only the 180 isoform demonstrated that the 180 isoform was required at adult synapses to maintain effective transmission with repetitive stimulation whereas the 140 and/or 120 isoform(s) were sufficient to mediate the downregulation of synaptic vesicle cycling along the axon after synapse formation. However, the expression and targeting of each isoform and its relationship to distinct forms of synaptic vesicle cycling before and after synapse formation was previously unknown. By transfecting chick motoneurons with fluorescently tagged mouse 180, 140 and 120 isoforms, we show that before myotube contact the 180 and 140 isoforms are expressed in distinct puncta along the axon which are sites of an immature form (Brefeldin A sensitive, L-type Ca2+ channel mediated) of vesicle cycling. After myotube contact the 140 and 180 isoforms are downregulated from the axon and selectively targeted to the presynaptic terminal. This coincided with the downregulation of vesicle cycling along the axon and the expression of the mature form (BFA insensitive, P/Q type Ca2+ channel mediated) of vesicle cycling at the terminal. The synaptic targeting of exogenously expressed 180 and 140 isoforms also occurred when chick motoneurons contacted +/+ mouse myotubes; however only the 180 but not the 140 isoform was targeted on contact with NCAM−/− myotubes. These observations indicate that postsynaptic NCAM is required for the synaptic targeting of presynaptic 140 NCAM but that the localization of presynaptic 180 NCAM occurs via a different mechanism.

Frequency-Dependent Modes of Synaptic Vesicle Endocytosis and Exocytosis at Adult Mouse Neuromuscular Junctions

During locomotion, adult rodent lumbar motoneurons fire in high-frequency (80–100 Hz) 1–2 s bursts every several seconds, releasing between 10,000 and 20,000 vesicles per burst. The estimated total vesicle pool size indicates that all vesicles would be used within 30 s; thus, a mechanism for rapid endocytosis and vesicle recycling is necessary to maintain effective transmission and motor behavior. However, whether such rapid recycling exists at mouse neuromuscular junctions (NMJs) or how it is regulated has been unclear. Here, we show that much less FM1-43 dye is lost per stimulus with 100 Hz stimulation than with 10 Hz stimulation even when the same number of vesicles undergo exocytosis. Electrophysiological data using folimycin show this lesser amount of dye loss is caused in part by the rapid reuse of vesicles. We showed previously that a myosin light chain kinase (MLCK)–myosin II pathway was required for effective transmission at 100 Hz. Here, we confirm the activation of MLCK, based on increased nerve terminal phospho-MLC immunostaining, with 100 Hz but not with 10 Hz stimulation. We further demonstrate that activation of MLCK, by increased extracellular Ca2+, by PKC (protein kinase C) activation, or by a MLCK agonist peptide, reduces the amount of dye lost even with 10 Hz stimulation. MLCK activation at 10 Hz also resulted in more vesicles being rapidly reused. Thus, MLCK activation by 100 Hz stimulation switches the mechanism of vesicle cycling to a rapid-reuse mode and is required to sustain effective transmission in adult mouse NMJs.

Femtogram-level detection of Clostridium botulinum neurotoxin type A by sandwich immunoassay using nanoporous substrate and ultra-bright fluorescent suprananoparticles

We report a simple, robust fluorescence biosensor for the ultra-sensitive detection of Clostridium botulinum Neurotoxin Type A (BoNT/A) in complex, real-world media. High intrinsic signal amplification was achieved through the combined use of ultra-bright, photostable dye-doped nanoparticle (DOSNP) tags and high surface area nanoporous organosilicate (NPO) thin films. DOSNP with 22 nm diameter were synthesized with more than 200 times equivalent free dye fluorescence and conjugated to antibodies with average degree of substitution of 90 dyes per antibody, representing an order of magnitude increase compared with conventional dye-labeled antibodies. The NPO films were engineered to form constructive interference at the surface where fluorophores were located. In addition, DOSNP-labeled antibodies with NPO films increased surface roughness causing diffuse scattering resulting in 24% more scattering intensity than dye-labeled antibody with NPO films. These substrates were used for immobilization of capture antibodies against BoNT/A, which was further quantified by DOSNP-labeled signal antibodies. The combination of optical effects enhanced the fluorescence and, therefore, the signal-to-noise ratio significantly. BoNT/A was detected in PBS buffer down to 21.3 fg mL(-1) in 4 h. The assay was then extended to several complex media and the four-hour detection limit was found to be 145.8 fg mL(-1) in orange juice and 164.2 fg mL(-1) in tap water, respectively, demonstrating at least two orders of magnitude improvement comparing to the reported detection limit of other enzyme-linked immunosorbent assays (ELISA). This assay, therefore, demonstrates a novel method for rapid, ultra-low level detection of not only BoNT/A, but other analytes as well.

An activity-dependent increased role for L-type calcium channels in exocytosis is regulated by adrenergic signaling in chromaffin cells.

Chromaffin cells of the adrenal medulla represent a primary output of the sympathetic nervous system. Their electrical stimulation evokes the fusion of large dense core granules with the cell membrane and the exocytic release of multiple transmitter molecules into the circulation. There the transmitters contribute to the regulation of basic metabolism of the organism. Under physiological activity, granule fusion and transmitter release are limited by activity-dependent Ca(2+) influx, entering through multiple isoforms of voltage-gated calcium channels. In this study we utilize perforated-patch voltage-clamp recordings and depolarize mouse chromaffin cells in situ with action potential-like waveforms to mimic physiological firing. We measure calcium influx through specific isoforms and measure cell capacitance as an index of granule fusion. Combining these approaches we calculate specific stimulus-secretion efficiencies for L-type, N-type, P/Q-type and R-type calcium channels under varied physiological activity levels. Current influx through all channel subtypes exhibited an activity-dependent depression. As expected P/Q-type channels, while responsible for modest Ca(2+) influx, are tightly coupled to catecholamine secretion under all conditions. We further find that stimulation designed to match sympathetic input under the acute stress response recruits L-type channels to a state of enhanced stimulus-secretion efficiency. N- and R-type channels do not undergo activity-dependent recruitment and remain loosely coupled to the secretion. Thus, only L-type channels exhibit activity-dependent changes in their stimulus-secretion function under physiological stimulation. Lastly, we show that treatment with the beta-adrenergic agonist, isoproterenol, specifically blocks the increase in the stimulus-secretion function of L-type channels. Thus, increased cell firing specifically enhances stimulus-secretion coupling of L-type Ca(2+) channels in chromaffin cells in situ. This mechanism is regulated by an adrenergic signaling pathway.