Cholesterol-Independent Effects of Methyl-β-Cyclodextrin on Chemical Synapses

The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14°C but not from those acclimatized to 21°C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation

The role of phospholipase D in regulated exocytosis

Background: Phospholipase D (PLD)-derived phosphatidic acid (PA) is suggested to function in exocytosis. Results: PLD activity on the plasma membrane was higher than on vesicles. PLD inhibitors suppressed some fusion parameters, but blockade of PA did not affect fusion. Conclusion: PLD-derived PA has modulatory rather than direct effects on fusion. Significance: Identifying critical mechanistic components may enable interventions to target diseases affecting exocytosis

Anionic lipids in Ca²+ -triggered fusion

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca 2+-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca 2+-sensing and even bind Ca 2+ directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca 2+ sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined

Identifying critical components of native Ca²⁺-triggered membrane fusion : integrating studies of proteins and lipids

Ca2+-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca2+-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayermerger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca2+ sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca2+ sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca2+ sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca2+-triggered membrane fusion

Cholesterol content, depletion, and recovery differ significantly between cold- and warm-acclimatized groups: comparison of isolated nerve and muscle.

<p>Cholesterol concentrations were assessed independently in both isolated nerve and muscle tissue. Lipids were extracted separately from isolated nervous tissue (ventral nerve cords from the crayfish abdomen) and isolated muscles. Nerve (left): initial concentrations of cholesterol were significantly reduced in warm- vs. cold-acclimatized animals. Cold-acclimatized group (open bars). Application of 10 mM MβCD caused a significant reduction in cholesterol after 10 min. Attempts to recover cholesterol by perfusion with Ch-HPβCD (recovery) and a saline wash were unsuccessful. Warm-acclimatized group (solid bars). Application of 10 mM MßCD also caused a significant reduction in cholesterol after 10 min. Subsequent perfusion with Ch-HPβCD and saline resulted in recovery of cholesterol to initial levels (i.e. baseline). Muscle (right): initial cholesterol levels were comparable in both the cold- and warm-acclimatized groups. Application of 10 mM MβCD caused a significant reduction in cholesterol after 10 min regardless of the acclimatization condition. Again, attempts to recover cholesterol using Ch-HPβCD and a saline wash were unsuccessful in the cold-acclimatized group. Notably, in the warm-acclimatized group, 3 min exposure to MβCD already resulted in a significant reduction of muscle cholesterol. Perfusion with Ch-HPβCD and saline was sufficient to return cholesterol levels to initial (i.e. baseline) levels in the warm-acclimatized group. N = 7.* indicates P<0.05</p

Chemical composition of salines used. All values are expressed in mM.

<p>Chemical composition of salines used. All values are expressed in mM.</p

MβCD alters postsynaptic responsiveness.

<p>Intracellular voltage recordings were made from L1 muscle fibers within segment IV while iontophoretically applying L-glutamate and simultaneously injecting hyperpolarizing current intracellularly. These recordings detected changes in input resistance of muscle fibers as well as any changes in postsynaptic receptor sensitivity. Under no conditions was there an effect on the input resistance of muscle fibers (open and closed squares). <i>A</i>. Cold-acclimatized group (open symbols). Responsiveness to iontophoretically applied glutamate (open circles) revealed a significant (P<0.05) transient increase following the application of 10 mM MβCD, that subsequently resulted in a 17%/min reduction in the sensitivity to applied glutamate which plateaued within 6 min. Attempts to recover responsiveness, using a cholesterol-loaded cyclodextrin (Ch-HPβCD) and a saline wash, were unsuccessful. <i>B</i>. Warm-acclimatized group (closed symbols). While there was no immediate change in responsiveness to applied glutamate (closed circles), a subsequent gradual reduction during exposure to MβCD occurred. Application of Ch-HPβCD recovered responsiveness to 90% of initial values, and a subsequent saline wash resulted in a return to control values. <i>C-D</i>. Control recordings from cold- and warm-acclimatized groups were stable throughout the 40min recording period. Insets: recordings of input resistance and responsiveness to iontophoretically applied glutamate at selected time points. * indicates P<0.05, † indicates P<0.01. N = 7. Scale bars: 15 mV, 250 ms.</p

MβCD caused comparable significant cholesterol depletion in warm- and cold-acclimatized groups but this effect was irreversible in the latter.

<p>Cholesterol concentrations were assessed in the same neuromuscular preparations used for the electrophysiological analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036395#pone-0036395-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036395#pone-0036395-g002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036395#pone-0036395-g003" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036395#pone-0036395-g004" target="_blank">4</a>). Cold-acclimatized groups (open bars). Application of 10 mM MβCD resulted in a significant reduction in cholesterol after 10 min. Attempts to recover cholesterol by perfusion with Ch-HPβCD (RECOVERY) were unsuccessful. Warm-acclimatized groups (solid bars). Application of 10 mM MβCD resulted in a significant reduction in cholesterol after 10 min, comparable to that measured in the cold-acclimatized group. Subsequent perfusion with Ch-HPβCD resulted in recovery to a level comparable to the saline control group (i.e. baseline). N = 7. * indicates P<0.05.</p

Input resistances in deep abdominal extensor muscle fibers, estimated using current pulses of varying amplitude (i.e. “slope” resistance).

<p>Input resistance was measured in the same fibers in 23% Ca²⁺ Saline (“Control”) and, subsequently, in the presence of 10 mM MβCD (N = 5 in all cases).</p

MβCD causes a reduction in EJPs which coincide with impulse failure only in neuromuscular preparations from cold-acclimatized animals.

<p>Intracellular recordings were made from L1 muscle fibers within the 4^th abdominal segment while simultaneously recording extracellularily through a loose patch electrode placed over a synapse. These recordings detect the propagating action potential (AP) invading the presynaptic terminal, the postsynaptic current following the AP, and an EJP. <i>A</i>. Cold-acclimatized group (open symbols). Control (no MβCD, open circles) depicts low-frequency depression, inherent during phasic stimulation. Application of 10 mM MβCD resulted in an initial significant (P<0.05) transient increase in EJP amplitude followed by a rapid reduction in EJP amplitude (P<0.05) which did not recover following the application of 10mM Ch-HPβCD or a subsequent saline wash. <i>B</i>. Warm-acclimatized group (closed symbols). Application of 10 mM MβCD resulted in an initial significant (P<0.05) transient increase in EJP amplitude, which quickly returned to control values, and remained stable for the remainder of the recording period. Inset (A–B) shows recorded signals of EJPs at selected time points. Scale bars: 5 mV, 5 ms. <i>C</i>. Extracellularily recorded nerve terminal currents from cold-acclimatized animals. The nerve terminal current remained stable during the pre-application period; application of 10 mM MβCD resulted in a loss of the nerve terminal signal in 6/7 trials, consequently producing a marked reduction in the amplitude of the nerve terminal current (P<0.05) which did not recover with the application of 10 mM Ch-HPβCD or a subsequent saline wash. <i>D</i>. Extracellularily recorded nerve terminal currents from warm-acclimatized animals. Application of 10 mM MβCD had no effect on the amplitude of the nerve terminal current. Inset (C–D) shows traces from loose patch recordings at selected time points. Initial downward deflection is the nerve terminal current, following by the post-synaptic current. Scale bars: 0.5 nA, 5ms. N = 7. * indicates P<0.05, † indicates P<0.01.</p