Isoform specificity of PKMs during long-term facilitation in Aplysia is mediated through stabilization by KIBRA

Abstract

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems including the hermaphrodite marine mollusk, Aplysia californica. Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform was required, the kidney and brain expressed adaptor protein (KIBRA) was also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant negative (DN)-PKMs to erase LTF is correlated with isoform specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an alpha-helix 'handle' in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia. 3 Significance StatementLong lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs in not isoform specific, isoform specificity is defined by a 'handle' helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

KIBRA Regulates AMPA Receptor Expression, Synaptic Plasticity, and Memory in an Age-Dependent Manner

“…Thus, a shift in the balance of endosomal trafficking of AMPARs toward lysosomes can alter AMPAR proteostasis and availability of these channels for synaptic insertion. We find that KIBRA is required to maintain extrasynaptic pools of AMPARs in adult neurons, consistent with prior studies demonstrating that KIBRA can stabilize its binding partners against degradation (Xiao et al, 2011;Vogt-Eisele et al, 2014;Hu et al, 2017;Ferguson et al, 2019;Song et al, 2019). Interestingly, we also see a reduction in the KIBRA homolog WWC2 in adult KIBRA cKO neurons.…”

“…3A,B) supporting a role for KIBRA in AMPAR proteostasis. Consistent with prior work, we also observe a reduction in PKMζ (WT, 100 ± 2%; cKO 74 ± 2%, p < 0.001), a brain-specific kinase that interacts with KIBRA (Buther et al, 2004;Vogt-Eisele et al, 2014;Ferguson et al, 2019). Acute reduction of KIBRA also decreased the basal expression of KIBRA homolog and binding partner WWC2 (Fig.…”

“…Neurons maintain distinct pools of AMPARs at excitatory synapses, including a synaptically localized pool which mediates neuronal communication, as well as extra- synaptic surface and intracellular pools that can be rapidly mobilized and inserted into the synapse to facilitate synaptic potentiation (Penn et al, 2017). KIBRA forms a complex with AMPARs (Makuch et al, 2011; Heitz et al, 2016) and has been shown to prevent degradation of multiple binding partners (Xiao et al, 2011; Vogt-Eisele et al, 2014; Hu et al, 2017; Ferguson et al, 2019; Song et al, 2019), thus we hypothesized that loss of KIBRA disrupts AMPAR proteostasis and depletes AMPARs from extrasynaptic pools required for LTP. To test this hypothesis, we performed subcellular fractionation on hippocampal area CA1 tissue from KIBRA cKO and WT mice and examined total, membrane-localized, and synaptically-localized (postsynaptic density- associated) AMPARs (Chiu et al, 2017).…”

“…Additionally, loss of KIBRA reduces the basal expression of multiple binding partners including PKMζ (Vogt-Eisele et al, 2014; Hu et al, 2017; Ferguson et al, 2019), Lats1/2 (Xiao et al, 2011), and Rab27a (Song et al, 2019). However, it is currently unclear if KIBRA regulates AMPA proteostasis, or if KIBRA’s regulation of AMPA receptors changes with age.…”

Functional characterization of a neuropeptide receptor exogenously expressed in Aplysia neurons

“…Initially, pNEX was constructed with the AK01a gene (shaker K + channel), and microinjected into Aplysia neurons, which demonstrated that the AK01a channel could modulate the firing of the injected neuron and regulate synaptic interactions [61]. Subsequent studies used pNEX to overexpress the target proteins and elucidated molecular mechanisms of synaptic plasticity [62][63][64][65][66][67][68]. Notably, small molecule neurotransmitter receptors (1ɑ metabotropic glutamate receptor [69]) and biogenic amine receptors (octopamine receptor) [64]), which are G-protein coupled receptors, were also expressed in Aplysia neurons through the vector pNEX.…”