Cell-Specific PKM Isoforms Contribute to the Maintenance of Different Forms of Persistent Long-Term Synaptic Plasticity.

Multiple kinase activations contribute to long-term synaptic plasticity, a cellular mechanism mediating long-term memory. The sensorimotor synapse of Aplysia expresses different forms of long-term facilitation (LTF)-nonassociative and associative LTF-that require the timely activation of kinases, including protein kinase C (PKC). It is not known which PKC isoforms in the sensory neuron or motor neuron L7 are required to sustain each form of LTF. We show that different PKMs, the constitutively active isoforms of PKCs generated by calpain cleavage, in the sensory neuron and L7 are required to maintain each form of LTF. Different PKMs or calpain isoforms were blocked by overexpressing specific dominant-negative constructs in either presynaptic or postsynaptic neurons. Blocking either PKM Apl I in L7, or PKM Apl II or PKM Apl III in the sensory neuron 2 d after 5-hydroxytryptamine (5-HT) treatment reversed persistent nonassociative LTF. In contrast, blocking either PKM Apl II or PKM Apl III in L7, or PKM Apl II in the sensory neuron 2 d after paired stimuli reversed persistent associative LTF. Blocking either classical calpain or atypical small optic lobe (SOL) calpain 2 d after 5-HT treatment or paired stimuli did not disrupt the maintenance of persistent LTF. Soon after 5-HT treatment or paired stimuli, however, blocking classical calpain inhibited the expression of persistent associative LTF, while blocking SOL calpain inhibited the expression of persistent nonassociative LTF. Our data suggest that different stimuli activate different calpains that generate specific sets of PKMs in each neuron whose constitutive activities sustain long-term synaptic plasticity.SIGNIFICANCE STATEMENT Persistent synaptic plasticity contributes to the maintenance of long-term memory. Although various kinases such as protein kinase C (PKC) contribute to the expression of long-term plasticity, little is known about how constitutive activation of specific kinase isoforms sustains long-term plasticity. This study provides evidence that the cell-specific activities of different PKM isoforms generated from PKCs by calpain-mediated cleavage maintain two forms of persistent synaptic plasticity, which are the cellular analogs of two forms of long-term memory. Moreover, we found that the activation of specific calpains depends on the features of the stimuli evoking the different forms of synaptic plasticity. Given the recent controversy over the role of PKMζ maintaining memory, these findings are significant in identifying roles of multiple PKMs in the retention of memory.

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia.

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

The atypical protein kinase C in Aplysia can form a protein kinase M by cleavage.

In vertebrates, a brain-specific transcript from the atypical protein kinase C (PKC) zeta gene encodes protein kinase M (PKM) zeta, a constitutively active kinase implicated in the maintenance of synaptic plasticity and memory. We have cloned the atypical PKC from Aplysia, PKC Apl III. We did not find a transcript in Aplysia encoding PKMzeta, and evolutionary analysis of atypical PKCs suggests formation of this transcript is restricted to vertebrates. Instead, over-expression of PKC Apl III in Aplysia sensory neurons leads to production of a PKM fragment of PKC Apl III. This cleavage was induced by calcium and blocked by calpain inhibitors. Moreover, nervous system enriched spliced forms of PKC Apl III show enhanced cleavage. PKC Apl III could also be activated through phosphorylation downstream of phosphoinositide 3-kinase. We suggest that PKM forms of atypical PKCs play a conserved role in memory formation, but the mechanism of formation of these kinases has changed over evolution.

HMWMAP2: new perspectives on a pathway to dendritic identity.

Neuronal polarity is established by the differentiation of two types of cytoplasmic processes: dendrites and the axon. These processes can be distinguished by their composition in microtubule-associated proteins, the high molecular weight MAP2 proteins (HMWMAP2) being found in the dendrites and tau proteins in the axon. It is believed that the main contribution of HMWMAP2 to the acquisition and maintenance of dendrites is to promote microtubule assembly and stability. However, recent studies force us to enlarge our view on how HMWMAP2 might contribute to defining the role of the dendritic microtubules. The purpose of this article is to convey our view that HMWMAP2 are important players in defining the contribution of microtubules to dendritic identity by anchoring membranous organelles and signaling proteins to the dendritic microtubules and by being a receptor for neurosteroids.

Tau interacts with Golgi membranes and mediates their association with microtubules.

Tau, a microtubule-associated protein enriched in the axon, is known to stabilize and promote the formation of microtubules during axonal outgrowth. Several studies have reported that tau was associated with membranes. In the present study, we further characterized the interaction of tau with membranous elements by examining its distribution in subfractions enriched in either Golgi or endoplasmic reticulum membranes isolated from rat brain. A subfraction enriched with markers of the medial Golgi compartment, MG160 and mannosidase II, presented a high tau content indicating that tau was associated with these membranes. Electron microscope morphometry confirmed the enrichment of this subfraction with Golgi membranes. Double-immunogold labeling experiments conducted on this subfraction showed the direct association of tau with vesicles labeled with either an antibody directed against MG160 or TGN38. The association of tau with the Golgi membranes was further confirmed by immunoisolating Golgi membranes with an anti-tau antibody. Immunogold labeling confirmed the presence of tau on the Golgi membranes in neurons in vivo. Overexpression of human tau in primary hippocampal neurons induced the formation of large Golgi vesicles that were found in close vicinity to tau-containing microtubules. This suggested that tau could serve as a link between Golgi membranes and microtubules. Such role for tau was demonstrated in an in vitro reconstitution assay. Finally, our results showed that some tau isoforms present in the Golgi subfraction were phosphorylated at the sites recognized by the phosphorylation-dependent antibodies PHF-1 and AT-8.

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons.

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.

The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells.

The expression of microtubule-associated protein 2 (MAP2), developmentally regulated by alternative splicing, coincides with neurite outgrowth. MAP2 proteins contain a microtubule-binding domain (C-terminal) that promotes microtubule assembly and a poorly characterized domain, the projection domain (N-terminal), extending at the surface of microtubules. MAP2b differs from MAP2c by an additional sequence of 1372 amino acids in the projection domain. In this study, we examined the role of the projection domain in the protrusion of microtubules from the cell surface and the subsequent process formation in Sf9 cells. In this system, MAP2b has a lower capacity to induce process formation than MAP2c. To investigate the role of the projection domain in this event, we expressed truncated forms of MAP2b and MAP2c that have partial or complete deletion of their projection domain in Sf9 cells. Our results indicate that process formation is induced by the microtubule-binding domain of these MAP2 proteins and is regulated by their projection domain. Furthermore, the microtubule-binding activity of MAP2b and MAP2c truncated forms as well as the structural properties of the microtubule bundles induced by them do not seem to be the only determinants that control the protrusion of microtubules from the cell surface in Sf9 cells. Rather, our data suggest that microtubule protrusion and process formation are regulated by intramolecular interactions between the projection domain and its microtubule-binding domain in MAP2b.