Rescue of tau-induced synaptic transmission pathology by paclitaxel.

Behavioral and electrophysiological studies of Alzheimer's disease (AD) and other tauopathies have revealed that the onset of cognitive decline correlates better with synaptic dysfunctions than with hallmark pathologies such as extracellular amyloid-ß plaques, intracellular hyperphosphorylated tau or neuronal loss. Recent experiments have also demonstrated that anti-cancer microtubule (MT)-stabilizing drugs can rescue tau-induced behavioral decline and hallmark neuron pathologies. Nevertheless, the mechanisms underlying tau-induced synaptic dysfunction as well as those involved in the rescue of cognitive decline by MTs-stabilizing drugs remain unclear. Here we began to study these mechanisms using the glutaminergic sensory-motoneuron synapse derived from Aplysia ganglia, electrophysiological methods, the expression of mutant-human tau (mt-htau) either pre or postsynaptically and the antimitotic drug paclitaxel. Expression of mt-htau in the presynaptic neurons led to reduced excitatory postsynaptic potential (EPSP) amplitude generated by rested synapses within 3 days of mt-htau expression, and to deeper levels of homosynaptic depression. mt-htau-induced synaptic weakening correlated with reduced releasable presynaptic vesicle pools as revealed by the induction of asynchronous neurotransmitter release by hypertonic sucrose solution. Paclitaxel totally rescued tau-induced synaptic weakening by maintaining the availability of the presynaptic vesicle stores. Postsynaptic expression of mt-htau did not impair the above described synaptic-transmission parameters for up to 5 days. Along with earlier confocal microscope observations from our laboratory, these findings suggest that tau-induced synaptic dysfunction is the outcome of impaired axoplasmic transport and the ensuing reduction in the releasable presynaptic vesicle stores rather than the direct effects of mt-htau or paclitaxel on the synaptic release mechanisms.

Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy.

In differentiated axons almost all microtubules (MTs) uniformly point their plus ends towards the axonal tip. The uniform polar pattern provides the structural substrate for efficient organelle transport along axons. It is generally believed that the mass and pattern of MTs polar orientation remain unchanged in differentiated neurons. Here we examined long-term effects of the MTs stabilizing reagent paclitaxel (taxol) over MTs polar orientation and organelle transport in cultured Aplysia neurons. Unexpectedly, we found that rather than stabilizing the MTs, paclitaxel leads to their massive polar reconfiguration, accompanied by impaired organelle transport. Washout of paclitaxel does not lead to recovery of the polar orientation indicating that the new pattern is self-maintained. Taken together the data suggest that MTs in differentiated neurons maintain the potential to be reconfigured. Such reconfiguration may serve physiological functions or lead to degeneration. In addition, our observations offer a novel mechanism that could account for the development of peripheral neuropathy in patients receiving paclitaxel as an antitumor drug.

Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices.

Interfacing neurons with micro- and nano-electronic devices has been a subject of intense study over the last decade. One of the major problems in assembling efficient neuro-electronic hybrid systems is the weak electrical coupling between the components. This is mainly attributed to the fundamental property of living cells to form and maintain an extracellular cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by propagating action potentials and thus reduces the signal-to-noise ratio. Reducing the cleft thickness, and thereby increasing the seal resistance formed between the neurons and the sensing surface, is thus a challenge and could improve the electrical coupling coefficient. Using electron microscopic analysis and field potential recordings, we examined here the use of gold micro-structures that mimic dendritic spines in their shape and dimensions to improve the adhesion and electrical coupling between neurons and micro-electronic devices. We found that neurons cultured on a gold-spine matrix, functionalized by a cysteine-terminated peptide with a number of RGD repeats, readily engulf the spines, forming tight apposition. The recorded field potentials of cultured Aplysia neurons are significantly larger using gold-spine electrodes in comparison with flat electrodes.

Activity-dependent calpain activation plays a critical role in synaptic facilitation and post-tetanic potentiation.

Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured Aplysia neurons (B2 and MCn), we demonstrate here that the calcium-activated protease-calpain serves as a major regulating element in the cascade that links electrical activity, elevation of the free intracellular calcium concentration, and short-term synaptic enhancements such as facilitation and PTP. Our study revealed that calpain inhibitors (calpeptin and MG132) transform a facilitating synapse into a depressing one, and reduce its PTP by 80.6%. Inhibition of CaM kinases, PKA, and MAPK also reduced PTP at this synapse. When inhibitors of these kinases were applied together with calpeptin, tetanic stimuli led to synaptic depression. We concluded that at this synapse facilitation and PTP are mediated mainly by the calpain-dependent processes and to a smaller extent by the CaMKs/PKA/MAPK-dependent cascades.

Calpain inhibitors alter the excitable membrane properties of cultured aplysia neurons.

The calpain superfamily of calcium-dependent papain-like cysteine proteases constitutes highly conserved proteases that function to posttranslationally modify substrates by partial proteolysis. Calpains are known to proteolyze >100 substrates that lack strong sequence homology. Consequently, the calpain superfamily has been implicated in playing a central role in diverse physiological and pathological processes. Investigation of the physiological functions of calpains, on the one hand, and the need to develop pharmacological reagents to inhibit calpain-mediated pathological processes, on the other hand, led to the development of numerous calpain inhibitors. Using cultured Aplysia neurons and voltage-clamp analysis, we report here that the calpain inhibitors calpeptin, MG132, and the calpain inhibitor XII inhibit voltage-gated potassium conductance and moderately reduce the sodium conductance. These consequently lead to spike broadening and increased calcium influx. Such alterations of the excitable membrane properties may alter the normal patterns of neuronal and muscle electrical activities and thus should be taken into account when evaluating the effects of calpain inhibitors as protective/therapeutic drugs and as research tools.

Calcium-activated proteases are critical for refilling depleted vesicle stores in cultured sensory-motor synapses of Aplysia.

Aplysia motoneurons cocultured with a presynaptic sensory neuron exhibit homosynaptic depression when stimulated at low frequencies. A single bath application of serotonin (5HT) leads within seconds to facilitation of the depressed synapse. The facilitation is attributed to mobilization of neurotransmitter-containing vesicles from a feeding vesicle store to the depleted, readily releasable pool by protein kinase C (PKC). Here, we demonstrate that the calpain inhibitors, calpeptin, MG132, and ALLN, but not the proteasome inhibitors, lactacystin and clasto-lactacystin beta-lactone, block 5HT-induced facilitation of depressed synapses. Likewise the 5HT-induced enhancement of spontaneous miniature potentials (mEPSPs) frequency of depressed synapses is significantly reduced by calpeptin. In contrast, neither the facilitation of nondepressed synapses nor the enhancement of their mEPSPs frequency is affected by the inhibitor. The data suggest that action potentials-induced calcium influx activate calpains. These, in turn, play a role in the refilling processes of the depleted, releasable vesicle store.

Short window of opportunity for calpain induced growth cone formation after axotomy of Aplysia neurons.

Our laboratory has established that local activation of calpain by a transient elevation of the free intracellular calcium concentration is crucial for the induction of growth cone (GC) formation in cultured Aplysia neurons. The mechanisms and stages in which calpain is involved in the formation of a GC are not known. We began to study these questions by determining the nature of calpain's action and the stages in which calpain activity affects the cascade of events that leads to the formation of the GC and its extension. We report that the calpain-dependent transformation of an axonal segment into a GC occurs within a narrow window of opportunity that lasts approximately 5 min. If calpain is inhibited during this window of opportunity, GC formation does not occur. Inhibition of calpain after the window of opportunity slows down the rate of lamellipodial extension but doesn't arrest it. The proteolysis of spectrin, a calpain substrate and a major component of the membrane skeleton, occurs within this window of opportunity, in agreement with the hypothesis that spectrin proteolysis is an early step in the formation of the GC. If the onset of proteolysis is deferred, spectrin remains unchanged and GC formation is compromised. We suggest that calpain participates in two different processes: it is critical for the triggering of GC formation and plays a modulatory role during the extension of the GC's lamellipodia.