KIBRA anchoring the action of PKMζ maintains the persistence of memory.

How can short-lived molecules selectively maintain the potentiation of activated synapses to sustain long-term memory? Here, we find kidney and brain expressed adaptor protein (KIBRA), a postsynaptic scaffolding protein genetically linked to human memory performance, complexes with protein kinase Mzeta (PKMζ), anchoring the kinase's potentiating action to maintain late-phase long-term potentiation (late-LTP) at activated synapses. Two structurally distinct antagonists of KIBRA-PKMζ dimerization disrupt established late-LTP and long-term spatial memory, yet neither measurably affects basal synaptic transmission. Neither antagonist affects PKMζ-independent LTP or memory that are maintained by compensating PKCs in ζ-knockout mice; thus, both agents require PKMζ for their effect. KIBRA-PKMζ complexes maintain 1-month-old memory despite PKMζ turnover. Therefore, it is not PKMζ alone, nor KIBRA alone, but the continual interaction between the two that maintains late-LTP and long-term memory.

KIBRA repairs synaptic plasticity and promotes resilience to tauopathy-related memory loss.

Synaptic plasticity is obstructed by pathogenic tau in the brain, representing a key mechanism that underlies memory loss in Alzheimer's disease (AD) and related tauopathies. Here, we found that reduced levels of the memory-associated protein KIdney/BRAin (KIBRA) in the brain and increased KIBRA protein levels in cerebrospinal fluid are associated with cognitive impairment and pathological tau levels in disease. We next defined a mechanism for plasticity repair in vulnerable neurons using the C-terminus of the KIBRA protein (CT-KIBRA). We showed that CT-KIBRA restored plasticity and memory in transgenic mice expressing pathogenic human tau; however, CT-KIBRA did not alter tau levels or prevent tau-induced synapse loss. Instead, we found that CT-KIBRA stabilized the protein kinase Mζ (PKMζ) to maintain synaptic plasticity and memory despite tau-mediated pathogenesis. Thus, our results distinguished KIBRA both as a biomarker of synapse dysfunction and as the foundation for a synapse repair mechanism to reverse cognitive impairment in tauopathy.

KIBRA repairs synaptic plasticity and promotes resilience to tauopathy-related memory loss.

Synaptic plasticity is obstructed by pathogenic tau in the brain, representing a key mechanism that underlies memory loss in Alzheimer's disease (AD) and related tauopathies. Here, we define a mechanism for plasticity repair in vulnerable neurons using the C-terminus of the KIdney/BRAin (KIBRA) protein (CT-KIBRA). We show that CT-KIBRA restores plasticity and memory in transgenic mice expressing pathogenic human tau; however, CT-KIBRA did not alter tau levels or prevent tau-induced synapse loss. Instead, we find that CT-KIBRA binds to and stabilizes protein kinase Mζ (PKMζ) to maintain synaptic plasticity and memory despite tau-mediated pathogenesis. In humans we find that reduced KIBRA in brain and increased KIBRA in cerebrospinal fluid are associated with cognitive impairment and pathological tau levels in disease. Thus, our results distinguish KIBRA both as a novel biomarker of synapse dysfunction in AD and as the foundation for a synapse repair mechanism to reverse cognitive impairment in tauopathy.

Learning-induced ribosomal RNA is required for memory consolidation in mice-Evidence of differentially expressed rRNA variants in learning and memory.

The transition from short-term to long-term forms of synaptic plasticity requires protein synthesis and new gene expression. Most efforts to understand experience-induced changes in neuronal gene expression have focused on the transcription products of RNA polymerase II-primarily mRNAs and the proteins they encode. We recently showed that nucleolar integrity and activity-dependent ribosomal RNA (rRNA) synthesis are essential for the maintenance of hippocampal long-term potentiation (LTP). Consequently, the synaptic plasticity and memory hypothesis predicts that nucleolar integrity and activity dependent rRNA synthesis would be required for Long-term memory (LTM). We tested this prediction using the hippocampus-dependent, Active Place Avoidance (APA) spatial memory task and found that training induces de novo rRNA synthesis in mouse dorsal hippocampus. This learning-induced increase in nucleolar activity and rRNA synthesis persists at least 24 h after training. In addition, intra-hippocampal injection of the Pol I specific inhibitor, CX-5461 prior to training, revealed that de novo rRNA synthesis is required for 24 h memory, but not for learning. Using qPCR to assess activity-dependent changes in gene expression, we found that of seven known rRNA expression variants (v-rRNAs), only one, v-rRNA IV, is significantly upregulated right after training. These data indicate that learning induced v-rRNAs are crucial for LTM, and constitute the first evidence that differential rRNA gene expression plays a role in memory.

Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control.

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5' untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.

Inhibition of Protein kinase Mzeta (PKMζ) in the mesolimbic system alters cocaine sensitization in rats.

Chronic cocaine use produces long-lasting changes in reward circuits that may underlie the transition from casual to compulsive patterns of drug use. Although strong neuroadaptations within the mesocorticolimbic system are known to occur, the specific role of these drug-induced plasticities on sensitization remains to be elucidated. Here we investigate whether PKMζ, a protein involved in maintaining long-term potentiation (LTP), plays a role in these cocaine-induced changes in synaptic strengthening. We performed whole-cell voltage clamp recordings of putative ventral tegmental area (VTA) dopamine (DA) cells 24 hours after five days of 15 mg/kg i.p. cocaine or isovolumetric saline injections. We observed that superfusion of 5µM ZIP (PKMζ inhibitory peptide) decreased AMPA currents and AMPA/NMDA ratios only in cocaine sensitized rats. In vivo ZIP microinfusions (10 nmol) into the VTA after cocaine sensitization decreased locomotor activity on a subsequent cocaine challenge only if given ZIP is given before the withdrawal period. On the other hand, ZIP microinfusions into the nucleus accumbens (NAc) core after a seven days withdrawal period disrupt the expression of locomotor sensitization. The present data provide a potentially relevant region, and time-specific PKMζ-dependent brain mechanism that enables sensitization. Our results support the vision that addiction involves a pathological learning process. They imply that if this synaptic strengthening is reversed, changes in the behavioral response may also be overturned.

Inhibition of protein kinase Mζ disrupts the stable spatial discharge of hippocampal place cells in a familiar environment.

It is widely held that spatial computations in the rodent hippocampus require the location-specific discharge of place cells that together form a stable cognitive map used to solve and perform spatial tasks. It is not known, however, if map stability requires persistent hippocampal synaptic strength changes that are vulnerable to blockade of protein kinase Mζ (PKMζ) phosphorylation activity, a manipulation that reverses hippocampal LTP and disrupts multiple forms of long-term memory. Here we report that acute intrahippocampal inhibition of PKMζ disrupts place cell activity in a familiar environment, where the map is expected to be stable. After this disruption, new, stable spatial firing patterns can later form, but the new and original maps are unrelated even though the rat is exposed to a constant environment. We therefore propose that the previously demonstrated erasure of stored spatial memory and the disruption of place cell firing are parallel effects of PKMζ blockade. We similarly propose that the known sparing of new spatial memory formation depends on the sparing of new map formation. On these bases, we argue that the loss of the map used to perform a practiced spatial task leads to behavioral performance deficits, and that synaptic plasticity maintained by PKMζ, which stabilizes the map, is essential for the proper expression of spatial memory.

Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase M synthesis and induce immediate preconditioning protection of rat hippocampal slices.

Anaesthetic preconditioning occurs when a volatile anaesthetic, such as sevoflurane, is administered before a hypoxic or ischaemic insult; this has been shown to improve neuronal recovery after the insult. We found that sevoflurane-induced preconditioning in the rat hippocampal slice enhances the hypoxic hyperpolarization of CA1 pyramidal neurons, delays and attenuates their hypoxic depolarization, and increases the number of neurons that recover their resting and action potentials after hypoxia. These altered electrophysiological effects and the improved recovery corresponded with an increase in the amount of a constitutively active, atypical protein kinase C isoform found in brain, protein kinase M zeta (PKMζ). A selective inhibitor of this kinase, zeta inhibitory peptide (ZIP), blocked the increase in the total amount of PKMζ protein and the amount of the activated form of this kinase, phospho-PKMζ (p-PKMζ); it also blocked the altered electrophysiological effects and the improved recovery. We found that both cycloheximide, a general protein synthesis inhibitor, and rapamycin, a selective inhibitor of the mTOR pathway for regulating protein synthesis, blocked the increase in p-PKMζ, the electrophysiological changes, and the improved recovery due to sevoflurane-induced preconditioning. Glibenclamide, a KATP channel blocker, when present only during the hypoxia, prevented the enhanced hyperpolarization, the delayed and attenuated hypoxic depolarization, and the improved recovery following sevoflurane-induced preconditioning. To examine the function of persistent PKMζ and KATP channel activity after the preconditioning was established, we administered 4% sevoflurane for 30 min and then discontinued it for 30 min before 10 min of hypoxia. When either tolbutamide, a KATP channel blocker, or ZIP were administered at least 15 min after the washout of sevoflurane, there was little recovery compared with sevoflurane alone. Thus, continuous KATP channel and PKMζ activity are required to maintain preconditioning protection. We conclude that sevoflurane induces activation of the mTOR pathway, increasing the new protein synthesis of PKMζ, which is constitutively phosphorylated to its active form, leading to an increased KATP channel-induced hyperpolarizaton. This hyperpolarization delays and attenuates the hypoxic depolarization, improving the recovery of neurons following hypoxia. Thus, sevoflurane acts via a metabotropic pathway to improve recovery following hypoxia.

JAK/STAT: the enigma within the mystery of NMDAR-LTD.

In this issue of Neuron, Nicolas et al. (2012) show that JAK2/STAT3 signaling, a canonical pathway for transmitting information from the cell membrane to the nucleus, is critical for NMDAR-LTD, even in the absence of new DNA transcription.

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain.

BACKGROUND: Chronic pain occurs when normally protective acute pain becomes pathologically persistent. We examined here whether an isoform of protein kinase C (PKC), PKMζ, that underlies long-term memory storage in various brain regions, also sustains nociceptive plasticity in spinal cord dorsal horn (SCDH) mediating persistent pain. RESULTS: Cutaneous injury or spinal stimulation produced persistent increases of PKMζ, but not other atypical PKCs in SCDH. Inhibiting spinal PKMζ, but not full-length PKCs, reversed plasticity-dependent persistent painful responses to hind paw formalin and secondary mechanical hypersensitivity and SCDH neuron sensitization after hind paw capsaicin, without affecting peripheral sensitization-dependent primary heat hypersensitivity after hind paw capsaicin. Inhibiting spinal PKMζ, but not full-length PKCs, also reversed mechanical hypersensitivity in the rat hind paw induced by spinal stimulation with intrathecal dihydroxyphenylglycine. Spinal PKMζ inhibition also alleviated allodynia 3 weeks after ischemic injury in rats with chronic post-ischemia pain (CPIP), at a point when allodynia depends on spinal changes. In contrast, spinal PKMζ inhibition did not affect allodynia in rats with chronic contriction injury (CCI) of the sciatic nerve, or CPIP rats early after ischemic injury, when allodynia depends on ongoing peripheral inputs. CONCLUSIONS: These results suggest spinal PKMζ is essential for the maintenance of persistent pain by sustaining spinal nociceptive plasticity.

BDNF facilitates L-LTP maintenance in the absence of protein synthesis through PKMζ.

Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ.

Postsynaptic degeneration as revealed by PSD-95 reduction occurs after advanced Aß and tau pathology in transgenic mouse models of Alzheimer's disease.

Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer's disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Aß deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Aß or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.

How does PKMζ maintain long-term memory?

Most of the molecular mechanisms contributing to long-term memory have been found to consolidate information within a brief time window after learning, but not to maintain information during memory storage. However, with the discovery that synaptic long-term potentiation is maintained by the persistently active protein kinase, protein kinase Mζ (PKMζ), a possible mechanism of memory storage has been identified. Recent research shows how PKMζ might perpetuate information both at synapses and during long-term memory.

PKMzeta inhibition reverses learning-induced increases in hippocampal synaptic strength and memory during trace eyeblink conditioning.

A leading candidate in the process of memory formation is hippocampal long-term potentiation (LTP), a persistent enhancement in synaptic strength evoked by the repetitive activation of excitatory synapses, either by experimental high-frequency stimulation (HFS) or, as recently shown, during actual learning. But are the molecular mechanisms for maintaining synaptic potentiation induced by HFS and by experience the same? Protein kinase Mzeta (PKMzeta), an autonomously active atypical protein kinase C isoform, plays a key role in the maintenance of LTP induced by tetanic stimulation and the storage of long-term memory. To test whether the persistent action of PKMzeta is necessary for the maintenance of synaptic potentiation induced after learning, the effects of ZIP (zeta inhibitory peptide), a PKMzeta inhibitor, on eyeblink-conditioned mice were studied. PKMzeta inhibition in the hippocampus disrupted both the correct retrieval of conditioned responses (CRs) and the experience-dependent persistent increase in synaptic strength observed at CA3-CA1 synapses. In addition, the effects of ZIP on the same associative test were examined when tetanic LTP was induced at the hippocampal CA3-CA1 synapse before conditioning. In this case, PKMzeta inhibition both reversed tetanic LTP and prevented the expected LTP-mediated deleterious effects on eyeblink conditioning. Thus, PKMzeta inhibition in the CA1 area is able to reverse both the expression of trace eyeblink conditioned memories and the underlying changes in CA3-CA1 synaptic strength, as well as the anterograde effects of LTP on associative learning.

Separating kindling and LTP: lessons from studies of PKM zeta in developing and adult rats.

The kindling model of temporal lobe epilepsy (TLE) and the memory model of long-term potentiation (LTP) may have common underlying mechanisms. This is evident by the demonstration that certain signaling molecules play a key role in both. Recently, a brain specific isoform of protein kinase C (PKMzeta) has been shown to play a significant role in both maintaining LTP and memory storage. We were interested in determining if this kinase had a crossover role in kindling-induced epileptogenesis. Using developing and adult rats we examined the role of PKMzeta in kindling. In developing (P15) rats we determined the effect of PKMzeta on retention of amygdala kindling and kindling rate by intra-amygdala administration of a selective PKMzeta antagonist, ZIP (10 nmol). In adult rats we examined the effect of PKMzeta inhibition, ZIP (10 nmol), on after discharge (AD) thresholds and kindling retention using rapid hippocampal kindling. Inhibition of PKMzeta by the antagonist ZIP did not affect kindling rate or retention in developing rats. In addition there was also no observed effect on AD thresholds and kindling retention in adult rats. Our results show that, despite the similarities between kindling and LTP in their induction, there is dissociation in the role that PKMzeta plays within the two in maintenance. This may be of importance in establishing a separation between the pathophysiological processes involved in sustaining kindling and the physiological mechanisms involved in maintaining LTP and memory storage.

Boundary conditions for the maintenance of memory by PKMzeta in neocortex.

We report here that ZIP, a selective inhibitor of the atypical protein kinase C isoform PKMzeta, abolishes very long-term conditioned taste aversion (CTA) associations in the insular cortex of the behaving rat, at least 3 mo after encoding. The effect of ZIP is not replicated by a general serine/threonine protein kinase inhibitor that is relatively ineffective toward PKMzeta, is independent of the intensity of training and the perceptual quality of the taste saccharin (conditioned stimulus, CS), and does not affect the ability of the insular cortex to re-encode the same specific CTA association again. The memory trace is, however, insensitive to ZIP during or immediately after training. This implies that the experience-dependent cellular plasticity mechanism targeted by ZIP is established following a brief time window after encoding, consistent with the standard period of cellular consolidation, but then, once established, does not consolidate further to gain immunity to the amnesic agent. Hence, we conclude that PKMzeta is not involved in short-term CTA memory, but is a critical component of the cortical machinery that stores long- and very long-term CTA memories.

PKMzeta maintains spatial, instrumental, and classically conditioned long-term memories.

How long-term memories are stored is a fundamental question in neuroscience. The first molecular mechanism for long-term memory storage in the brain was recently identified as the persistent action of protein kinase Mzeta (PKMzeta), an autonomously active atypical protein kinase C (PKC) isoform critical for the maintenance of long-term potentiation (LTP). PKMzeta maintains aversively conditioned associations, but what general form of information the kinase encodes in the brain is unknown. We first confirmed the specificity of the action of zeta inhibitory peptide (ZIP) by disrupting long-term memory for active place avoidance with chelerythrine, a second inhibitor of PKMzeta activity. We then examined, using ZIP, the effect of PKMzeta inhibition in dorsal hippocampus (DH) and basolateral amygdala (BLA) on retention of 1-d-old information acquired in the radial arm maze, water maze, inhibitory avoidance, and contextual and cued fear conditioning paradigms. In the DH, PKMzeta inhibition selectively disrupted retention of information for spatial reference, but not spatial working memory in the radial arm maze, and precise, but not coarse spatial information in the water maze. Thus retention of accurate spatial, but not procedural and contextual information required PKMzeta activity. Similarly, PKMzeta inhibition in the hippocampus did not affect contextual information after fear conditioning. In contrast, PKMzeta inhibition in the BLA impaired retention of classical conditioned stimulus-unconditioned stimulus (CS-US) associations for both contextual and auditory fear, as well as instrumentally conditioned inhibitory avoidance. PKMzeta inhibition had no effect on postshock freezing, indicating fear expression mediated by the BLA remained intact. Thus, persistent PKMzeta activity is a general mechanism for both appetitively and aversively motivated retention of specific, accurate learned information, but is not required for processing contextual, imprecise, or procedural information.

Protein kinase M zeta regulation of Na/K ATPase: a persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures.

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia-hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMzeta), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the alpha1 and alpha2 isoforms of the Na/K ATPase. Similarly, active PKMzeta levels were increased at 24 h, but not 2 h, after preconditioning. PKMzeta overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMzeta regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit alpha1, dihydroouabain to inhibit alpha2/3 and a zeta-pseudosubstrate peptide to inhibit PKMzeta. These experiments showed that PKMzeta regulated both the activity and surface expression of the alpha1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and zeta-pseudosubstrate peptide were used to determine if PKMzeta or the alpha1 and alpha2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMzeta levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMzeta and Na/K ATPase in ischemic preconditioning.

Atypical protein kinase C in neurodegenerative disease I: PKMzeta aggregates with limbic neurofibrillary tangles and AMPA receptors in Alzheimer disease.

Protein kinase Mzeta (PKMzeta), an atypical protein kinase C (PKC) isoform, plays a key role in the maintenance of long-term potentiation (LTP), a persistent enhancement of AMPA receptor-mediated synaptic transmission, as well as in the persistence of memory in Drosophila. Because memory impairment in Alzheimer disease (AD) has been attributed to disruption of synaptic plasticity, we investigated the expression and distribution of PKMzeta in this disorder. We found that PKMzeta accumulated in neurofibrillary tangles (NFTs), whereas conventional and novel PKC isoforms did not. Unlike tau, which is present in all NFTs regardless of location, PKMzeta was found in a subset of NFTs restricted to limbic or medial temporal lobe structures (i.e. hippocampal formation, entorhinal cortex, and amygdala), areas implicated in memory loss in AD. Interestingly, PKMzeta was not identified in any NFTs in control brains derived from 6 elderly individuals without known cognitive impairment. In medial temporal lobe structures in AD, PKMzeta also occurred within abnormal neurites expressing MAP2, GluR1 and GluR2 as well as in perisomatic granules expressing GluR1 and GluR2, suggesting that aggregation of PKMzeta disrupts glutamatergic synaptic transmission. Together, these findings suggest a link between PKMzeta-mediated synaptic plasticity and memory impairment in AD.