Neuroprotective versus tumorigenic protein kinase C activators.

Protein kinase C (PKC) activators possess potent neurotrophic and neuroprotective activity, thus indicating potential applications in treating neurodegenerative diseases, stroke and traumatic brain injury. Although some activators, such as bryostatin and gnidimacrin, have been tested as antitumor agents, others, such as phorbol esters, are potent tumor promoters. All PKC activators downregulate PKC at high concentrations and long application times. However, tumorigenic activators downregulate certain PKC isozymes, especially PKCdelta, more strongly. Tumorigenic activators possess unique structural features that could account for this difference. At concentrations that minimize PKC downregulation, PKC activators can improve long-term memory, reduce beta-amyloid levels, induce synaptogenesis, promote neuronal repair and inhibit cell proliferation. Intermittent, low concentrations of structurally specific, non-tumorigenic PKC activators, therefore, could offer therapeutic benefit for a variety of neurologic disorders.

PKC activator therapeutic for mild traumatic brain injury in mice.

Traumatic brain injury (TBI) is a frequent consequence of vehicle, sport and war related injuries. More than 90% of TBI patients suffer mild injury (mTBI). However, the pathologies underlying the disease are poorly understood and treatment modalities are limited. We report here that in mice, the potent PKC activator bryostatin1 protects against mTBI induced learning and memory deficits and reduction in pre-synaptic synaptophysin and post-synaptic spinophylin immunostaining. An effective treatment has to start within the first 8h after injury, and includes 5 × i.p. injections over a period of 14 days. The treatment is dose dependent. Exploring the effects of the repeated bryostatin1 treatment on the processing of the amyloid precursor protein, we found that the treatment induced an increase in the putative α-secretase ADAM10 and a reduction in ß-secretase activities. Both these effects could contribute towards a reduction in ß-amyloid production. These results suggest that bryostatin1 protects against mTBI cognitive and synaptic sequela by rescuing synapses, which is possibly mediated by an increase in ADAM10 and a decrease in BACE1 activity. Since bryostatin1 has already been extensively used in clinical trials as an anti-cancer drug, its potential as a remedy for the short- and long-term TBI sequelae is quite promising.

Motile statocyst cilia transmit rather than directly transduce mechanical stimuli.

We have investigated the role of motile cilia in mechanotransduction by statocysts of the nudibranch mollusk Hermissenda crassicornis. Movement of the cilia that experience the weight of statoconia causes increased variance of voltage noise and membrane depolarization of the statocyst hair cell. Two complementary approaches were used to immobilize the cilia. Vanadate anion was iontophoretically injected into hair cells. This reversible inhibitor of vibratile form and to assume a more classic, pliable beat pattern. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. When the intracellular vanadate concentration approached 10(-5) M, the cilia were arrested in an effective stroke against the cell membrane. The cell no longer depolarized upon gravitational or local mechanical stimulation. Rapid reversal of ciliary inhibition by norepinephrine or slow reversal with time restored both the voltage noise and depolarization response. Cilia were rendered rigid and upright by covalent cross-linkage of their membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4'-dithiobisphenyl azide. In the initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential and membrane resistance remained essentially constant. Mechanical stimulation of the rigid cilia, normal to the cell membrane, elicited a generator potential of the same amplitude but of greater duration than before treatment. Because cilia that are partially arrested by vanadate undergo increased bending, although the hair cell shows decreased noise, neither the axoneme nor the ciliary membrane proper would appear to be sites of direct transduction. In cells with beating but stiffened cilia, however, the voltage noise becomes amplified, implying an increased efficiency of transduction. We suggest that active but rigid flexure of the axoneme is involved in amplification and continuous signal detection. The basal insertion area is the most likely transduction site, being the terminal leverage point through which force is applied to the plasma membrane via the flexing ciliary shaft.

Calcium-mediated reduction of ionic currents: a biophysical memory trace.

Learning behavior similar to vertebrate classical conditioning was demonstrated for the mollusc Hermissenda crassicornis. Postsynaptic membrane changes within well-defined neural systems that mediate the learning play a casual role in recording the learned association for later recall. Specific ionic currents in neural tissue undergo transformations lasting days after associative training with physiologic stimuli. During acquisition the intracellular calcium increases; this increase is accompanied by specific potassium current reduction that lasts for days after conditioning. The increase of calcium enhances calmodulin-dependent phosphorylation of proteins that either regulate or are part of ion channels. These currents and the conditions that precede their transformation occur in many types of vertebrate neurons, and hence this biophysical basis of Hermissenda learning could have relevance for species other than the gastropod studied.

Voltage-dependent calcium and potassium ion conductances: a contingency mechanism for an associative learning model.

Persistent light-induced depolarization results from Ca2+ influx across a photoreceptor membrane. The marked dependence on potential of this Ca2+ influx and a Ca+-dependent K+ efflux accounts for enhancement of the light-induced depolarization when light is paired with rotation. A positive feedback cycle between light-induced depolarization and synaptic depolarization due to stimulus pairing can explain long-lasting behavioral changes produced by associative training but not control paradigms. The sensitivity of this Ca2+ influx to intracellular levels of adenosine 3'-5'-monophosphate suggests biochemical steps for this model of associative learning.

Membrane depolarization accumulates during acquisition of an associative behavioral change.

Long-lasting electrical changes of identified Hermissenda neurons, the type B photoreceptors, can account for concomitant associative behavioral changes. Depolarization of the type B cells after paired light and rotation accumulates (as monitored with intracellular electrodes) with reptition. This accumulation was specific to stimulus pairing (versus light alone or explicitly unpaired stimuli) and to the orientation of the nervous system with respect to the center of rotation; it provides a neural step in the acquisition of associative behavioral changes for gastropod mollusks and possibly other species.

A spatial-temporal model of cell activation.

A spatial-temporal model of calcium messenger function is proposed to account for sustained cellular responses to sustained stimuli, as well as for the persistent enhancement of cell responsiveness after removal of a stimulus, that is, cellular memory. According to this model, spatial separation of calcium function contributes to temporal separation of distinct phases of the cellular response. At different cellular sites, within successive temporal domains, the calcium messenger is generated by different mechanisms and has distinct molecular targets. In particular, prolonged cell activation is brought about by the interaction of calcium with another spatially confined messenger, diacylglycerol, to cause the association of protein kinase C with the plasma membrane. Activity of the membrane-associated protein kinase C is controlled by the rate of calcium cycling across the plasma membrane. In some instances, a single stimulus induces both protein kinase C activation and calcium cycling and thus causes prolonged activation; but in others, a close temporal association of distinct stimuli brings about cell activation via interaction of these intracellular messengers. Persistent enhancement of cell responsiveness after removal of stimuli is suggested to be due to the continued association, or anchoring, of protein kinase C to the membrane.

Neural organization predicts stimulus specificity for a retained associative behavioral change.

Paired, but not random, presentations of light and rotation produced long-term changes in Hermissenda's response to light. The nature of this change depended on the orientation of the animals with respect to the center of rotation and was predicted by known organizational features of Hermissenda's nervous system. When rotation that excited caudal hair cells was paired with light, a significant increase in response latency to test lights resulted. Rotation exciting cephalic hair cells when paired with light decreased the response latencies compared with latencies produced by random presentation of light and rotation.

Primary changes of membrane currents during retention of associative learning.

A single identified neuron was repeatedly isolated by axotomy from the central nervous system of the nudibranch mollusk Hermissenda crassicornis. An early voltage-dependent outward K+ current of this neuron was reduced and more rapidly inactivated for animals previously trained with paired but not randomized light and rotation. Since this current change can affect interneuron and motorneuron output via known synaptic pathways, it helps explain a long-lasting behavioral change that shows the defining features of vertebrate associative learning.

Associative behavioral modification in hermissenda: cellular correlates.

Three days of training consisting of trials of light paired with rotation produces a long-term modification of photopositive behavior in Hermissenda crassicornis. The behavioral modification depends on the temporal association of light and rotation. For animals that received light paired with rotation, significant increases in the spontaneous activity of type B photoreceptors were correlated with changes in photopositive behavior after training. A persistent tonic depolarization of type B photoreceptors can explain the cellular changes correlated with the long-term behavioral modification produced by the temporal association of light and rotation.

Retention of an associative behavioral change in Hermissenda.

The nudibranch mollusk Hermissenda crassicornis is normally attracted to a test light. Three days of training consisting of 50 trials per day of light paired with a rotational stimulus led to a significant increase, lasting for days, in the animal's response latency to enter a test light. The group that received light associated with rotation was significantly different from groups subjected to nonassociative control procedures. Modifications of well-known sensory networks may be related to a behavioral change that shares several operational features with associative learning.

Isolation of a G protein that is modified by learning and reduces potassium currents in Hermissenda.

In Hermissenda crassicornis conditioned to associate light and rotation, type B photoreceptor neurons exhibit pairing-specific decreases in the potassium currents IA and IK-Ca, which account for many of the behavioral changes elicited by associative conditioning. To determine which proteins are involved in storage of this memory, high-performance liquid chromatography was used to examine proteins from Hermissenda eyes. Conditioning-specific changes in four phosphoproteins were observed 24 hours after conditioning. One of these proteins, cp20, was purified to apparent homogeneity and found to be a G protein. When injected back into Hermissenda type B cells, cp20 reduced IK and IK-Ca in a manner indistinguishable from the reduction caused by conditioning, suggesting that this protein may play a crucial role in memory acquisition or retention.

Ca2+-dependent protein kinase injection in a photoreceptor mimics biophysical effects of associative learning.

Iontophoretic injection of phosphorylase kinase, a Ca2+-calmodulin-dependent protein kinase, increased input resistance, enhanced the long-lasting depolarization component of the light response, and reduced the early transient outward K+ current, IA, and the late K+ currents, IB, in type B photoreceptors of Hermissenda crassicornis in a Ca2+-dependent manner. Since behavioral and biophysical studies have shown that similar membrane changes persist after associative conditioning, these results suggest that Ca2+-dependent protein phosphorylation could mediate the long-term modulation of specific K+ channels as a step in the generation of a coditioned behavioral change.

Soluble beta-amyloid induction of Alzheimer's phenotype for human fibroblast K+ channels.

Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease--namely, the absence of a 113-picosiemen potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that depends on functional 113-picosiemen potassium channels, was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased [Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by 10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function to produce symptoms such as memory loss by a means other than plaque formation.

Membrane changes in a single photoreceptor cause associative learning in Hermissenda.

Single type B photoreceptors in intact, restrained Hermissenda were impaled with a microelectrode and exposed to either paired or unpaired presentations of light and depolarizing current to simulate natural stimulus effects during conditioning with light and rotation. Paired, but not unpaired, stimulus presentations produced cumulative depolarization and increased input resistance in type B cells. These membrane changes are similar to those observed after pairings of light and rotation are administered to either intact animals or isolated nervous systems or when light is paired with electrical stimulation of the vestibular system in isolated nervous systems. One and two days after treatment, pairing- and light-specific suppression of phototactic behavior was observed in recovered animals. These findings indicate that the membrane changes of type B cells produced by pairing light with current injections cause acquisition of the learned behavior.

Protein kinase injection reduces voltage-dependent potassium currents.

Intracellular iontophoretic injection of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase increased input resistance and decreased a delayed voltage-dependent K+ current of the type B photoreceptor in the nudibranch Hermissenda crassicornis to a greater extent than an early, rapidly inactivating K+ current (IA). This injection also enhanced the long-lasting depolarization of type B cells after a light step. These findings suggest the involvement of cyclic adenosine monophosphate-dependent phosphorylation in the differential regulation of photoreceptor K+ currents particularly during illumination. On the other hand, conditioning-induced changes in IA may also be regulated by a different type of phosphorylation (for example, Ca2+-dependent).

Acetylcholine: release from neural tissue and identification by pyrolysis--gas chromatography.

Gas chromatographic analysis showed acetylcholine to be the only choline ester released from innervated longitudinal muscle of the guinea pigileum by electrical stimulation. The amount of acetylcholine measured by gas chromatography agreed almost exactly with that measured by bioassay. Denervated longitudinal muscle produced no acetylcholine, and treatment of the muscle with tetrodotoxin markedly reduced acetylcholine output. The method permits the recovery and quantitation of amounts of acetylcholine as low as 5 nanograms in 5 milliliters of tissue perfusates.

Early enhancement of calcium currents by H-ras oncoproteins injected into Hermissenda neurons.

Influx of calcium through membrane channels is an important initial step in signal transduction of growth signals. Therefore, the effects of Ras protein injection on calcium currents across the soma membrane of an identified neuron of the snail Hermissenda were examined. With the use of these post-mitotic cells, a voltage-sensitive, inward calcium current was increased 10 to 20 minutes after Harvey-ras oncoproteins were injected. The effects of oncogenic Harvey ras p21 protein (v-Ras) occurred quickly and were sustained, whereas the effects of proto-oncogenic ras protein (c-Ras) were transient. This relative potency is consistent with the activities of these oncoproteins in stimulating cell proliferation. Thus, this calcium channel may be a target for Ras action.

Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus.

Activation of protein kinase C (PKC) can mimic the biophysical effects of associative learning on neurons. Furthermore, classical conditioning of the rabbit nictitating membrane (a form of associative learning) produces translocation of PKC activity from the cytosolic to the membrane compartments of the CA1 region of the hippocampus. Evidence is provided here for a significant change in the amount and distribution of PKC within the CA1 cell field of the rabbit hippocampus that is specific to learning. This change is seen at 1 day after learning as focal increments of [3H]phorbol-12,13-dibutyrate binding to PKC in computer-generated images produced from coronal autoradiographs of rabbit brain. In addition, 3 days after learning, the autoradiographs suggest a redistribution of PKC within CA1 from the cell soma to the dendrites.

Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade.

Synaptic changes that underlie associative learning and memory begin with temporally related activity of two or more independent synaptic inputs to common postsynaptic targets. In turn, temporally related molecular events regulate cytosolic Ca2+ during progressively longer-lasting time domains. Associative learning behaviors of living animals have been correlated with changes of neuronal voltage-dependent K+ currents, protein kinase C-mediated phosphorylation and synthesis of the Ca2+ and GTP-binding protein, calexcitin (CE),and increased expression of the Ca2+-releasing ryanodine receptor (type II). These molecular events, some of which have been found to be dysfunctional in Alzheimer's disease, provide means of altering dendritic excitability and thus synaptic efficacy during induction, consolidation and storage of associative memory. Apparently, such stages of behavioral learning correspond to sequential differences of Ca2+ signaling that could occur in spatially segregated dendritic compartments distributed across brain structures, such as the hippocampus.