MRI of Capn15 Knockout Mice and Analysis of Capn 15 Distribution Reveal Possible Roles in Brain Development and Plasticity.

The Small Optic Lobe (SOL) family of calpains are intracellular cysteine proteases that are expressed in the nervous system and play an important role in neuronal development in both Drosophila, where loss of this calpain leads to the eponymous small optic lobes, and in mouse and human, where loss of this calpain leads to eye anomalies. Some human individuals with biallelic variants in CAPN15 also have developmental delay and autism. However, neither the specific effect of the loss of the Capn15 protein on brain development nor the brain regions where this calpain is expressed in the adult is known.

Biallelic variants in the small optic lobe calpain CAPN15 are associated with congenital eye anomalies, deafness and other neurodevelopmental deficits.

Microphthalmia, coloboma and cataract are part of a spectrum of developmental eye disorders in humans affecting ~12 per 100 000 live births. Currently, variants in over 100 genes are known to underlie these conditions. However, at least 40% of affected individuals remain without a clinical genetic diagnosis, suggesting variants in additional genes may be responsible.

A role for Numb in Protein kinase M (PKM)-mediated increase in surface AMPA receptors during facilitation in Aplysia.

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength.

The zinc fingers of the small optic lobes calpain bind polyubiquitin.

The small optic lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al. 2017a).

Selective Erasure of Distinct Forms of Long-Term Synaptic Plasticity Underlying Different Forms of Memory in the Same Postsynaptic Neuron.

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses.

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 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.

Investigating the Potential Signaling Pathways That Regulate Activation of the Novel PKC Downstream of Serotonin in Aplysia.

Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT) underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons.

A PKM generated by calpain cleavage of a classical PKC is required for activity-dependent intermediate-term facilitation in the presynaptic sensory neuron of Aplysia.

Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that dominant-negative PKMs show isoform-specificity.

Bidirectional regulation of eEF2 phosphorylation controls synaptic plasticity by decoding neuronal activity patterns.

At the sensory-motor neuron synapse of Aplysia, either spaced or continuous (massed) exposure to serotonin (5-HT) induces a form of intermediate-term facilitation (ITF) that requires new protein synthesis but not gene transcription. However, spaced and massed ITF use distinct molecular mechanisms to maintain increased synaptic strength. Synapses activated by spaced applications of 5-HT generate an ITF that depends on persistent protein kinase A (PKA) activity, whereas an ITF produced by massed 5-HT depends on persistent protein kinase C (PKC) activity.

Synapse formation changes the rules for desensitization of PKC translocation in Aplysia.

Protein kinase Cs (PKCs) are activated by translocating from the cytoplasm to the membrane. We have previously shown that serotonin-mediated translocation of PKC to the plasma membrane in Aplysia sensory neurons was subject to desensitization, a decrease in the ability of serotonin to induce translocation after previous application of serotonin. In Aplysia, changes in the strength of the sensory-motor neuron synapse are important for behavioral sensitization and PKC regulates a number of important aspects of this form of synaptic plasticity.

Serotonin-induced cleavage of the atypical protein kinase C Apl III in Aplysia.

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III.

Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II.

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium.

Molecular determinants of the spacing effect.

Long-term memory formation is sensitive to the pattern of training sessions. Training distributed over time (spaced training) is superior at generating long-term memories than training presented with little or no rest interval (massed training). This spacing effect was observed in a range of organisms from invertebrates to humans.

The role of C2 domains in PKC signaling.

More than two decades ago, the discovery of the first C2 domain in conventional Protein Kinase Cs (cPKCs) and of its role as a calcium-binding motif began to shed light on the activation mechanism of this family of Serine/Threonine kinases which are involved in several critical signal transduction pathways. In this chapter, we review the current knowledge of the structure and the function of the different C2 domains in PKCs. The C2 domain of cPKCs is a calcium sensor and its calcium-dependent binding to phospholipids is crucial for kinase activation.

The rates of protein synthesis and degradation account for the differential response of neurons to spaced and massed training protocols.

The sensory-motor neuron synapse of Aplysia is an excellent model system for investigating the biochemical changes underlying memory formation. In this system, training that is separated by rest periods (spaced training) leads to persistent changes in synaptic strength that depend on biochemical pathways that are different from those that occur when the training lacks rest periods (massed training). Recently, we have shown that in isolated sensory neurons, applications of serotonin, the neurotransmitter implicated in inducing these synaptic changes during memory formation, lead to desensitization of the PKC Apl II response, in a manner that depends on the method of application (spaced versus massed).

Inhibitory responses in Aplysia pleural sensory neurons act to block excitability, transmitter release, and PKC Apl II activation.

Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons.

A new mechanism of action of a C2 domain-derived novel PKC inhibitor peptide.

Novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. We have previously shown that the Aplysia novel PKC Apl II's C2 domain inhibits binding of diacylglycerol (DAG) to the C1 domain and that this inhibition is removed by phosphatidic acid (PA) binding to the C1b domain. Another model for C2 domain regulation of nPKCs suggests that the C2 domain binds to receptors for activated C kinase (RACKs) to assist in kinase translocation and activation.

Live-imaging of PKC translocation in Sf9 cells and in aplysia sensory neurons.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent.

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.

PKC differentially translocates during spaced and massed training in Aplysia.

Learning is highly regulated by the pattern of training. In Aplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (5HT)-mediated activation of the novel PKC Apl II.

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.

Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCepsilon, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA.

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.

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.