Keiko Tanaka-Yamamoto

Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression.

BackgroundRegulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop.ResultsLTD in the cerebellum of Ptprr−/− mice is strongly impeded, in vitro and in vivo. Comparison of basal phospho-MAPK levels between wild-type and PTPRR deficient cerebellar slices revealed increased levels in mutants. This high basal phospho-MAPK level attenuated further increases in phospho-MAPK during chemical induction of LTD, essentially disrupting the positive feedback loop and preventing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation and endocytosis.ConclusionsOur findings indicate an important role for PTPRR in maintaining low basal MAPK activity in Purkinje cells. This creates an optimal ‘window’ to boost MAPK activity following signals that induce LTD, which can then propagate through feed-forward signals to cause AMPAR internalization and LTD.

Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors

Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2−/− mice, remains unexplored. Here we show that Shank2−/− mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2−/− mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2−/− mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

Functional and Physical Interaction of Diacylglycerol Kinase ζ with Protein Kinase Cα Is Required for Cerebellar Long-Term Depression

The balance between positive and negative regulators required for synaptic plasticity must be well organized at synapses. Protein kinase Cα (PKCα) is a major mediator that triggers long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the cerebellum. However, the precise mechanisms involved in PKCα regulation are not clearly understood. Here, we analyzed the role of diacylglycerol kinase ζ (DGKζ), a kinase that physically interacts with PKCα as well as postsynaptic density protein 95 (PSD-95) family proteins and functionally suppresses PKCα by metabolizing diacylglycerol (DAG), in the regulation of cerebellar LTD. In Purkinje cells of DGKζ-deficient mice, LTD was impaired and PKCα was less localized in dendrites and synapses. This impaired LTD was rescued by virus-driven expression of wild-type DGKζ, but not by a kinase-dead mutant DGKζ or a mutant lacking the ability to localize at synapses, indicating that both the kinase activity and synaptic anchoring functions of DGKζ are necessary for LTD. In addition, experiments using another DGKζ mutant and immunoprecipitation analysis revealed an inverse regulatory mechanism, in which PKCα phosphorylates, inactivates, and then is released from DGKζ, is required for LTD. These results indicate that DGKζ is localized to synapses, through its interaction with PSD-95 family proteins, to promote synaptic localization of PKCα, but maintains PKCα in a minimally activated state by suppressing local DAG until its activation and release from DGKζ during LTD. Such local and reciprocal regulation of positive and negative regulators may contribute to the fine-tuning of synaptic signaling. SIGNIFICANCE STATEMENT Many studies have identified signaling molecules that mediate long-term synaptic plasticity. In the basal state, the activities and concentrations of these signaling molecules must be maintained at low levels, yet be ready to be boosted, so that synapses can undergo synaptic plasticity only when they are stimulated. However, the mechanisms involved in creating such conditions are not well understood. Here, we show that diacylglycerol kinase ζ (DGKζ) creates optimal conditions for the induction of cerebellar long-term depression (LTD). DGKζ works by regulating localization and activity of protein kinase Cα (PKCα), an important mediator of LTD, so that PKCα effectively responds to the stimulation that triggers LTD.

Selective transgene expression in cerebellar Purkinje cells and granule cells using adeno-associated viruses together with specific promoters

Adeno-associated virus (AAV) is a powerful tool for gene delivery into the brain and has been used for transgene expression in the cerebellar cortex. Although the efficacies of different AAV serotypes to transduce cerebellar Purkinje cells were examined, it has been difficult to achieve cell-type specific transgene expression. Here we used AAV serotype 1 with two specific promoters, namely, Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) and the minimum region of the GABAA receptor α6 subunit (GABRα6) promoters, and compared their expression patterns in the cerebellar cortex with the expression patterns of ubiquitous promoters that are often used for AAV-mediated expression. Whereas AAV with ubiquitous promoters, the cytomegalovirus early enhancer/chicken β-actin promoter, and a small fragment of the synapsin-1 gene promoter caused ubiquitous expression in all cerebellar neurons tested, AAV with the CaMKIIα promoter injected into 10-day-old mice enabled selective expression in Purkinje cells. Furthermore, we developed AAV with the GABRα6 promoter, and succeeded for the first time to express the transgene exclusively in granule cells. Fresh cerebellar slices of mice injected with these AAVs were applicable for physiological experiments, such as patch clamp recording, optogenetic imaging, and stimulation. Thus, these AAV vectors are useful tools towards understanding the basic properties of cerebellar neurons or mechanisms of cerebellar functions. Particularly, selective expression in Purkinje or granule cells is useful for analyses using genetically-modified animals, such as knockout mice.

2013 Special Issue: Mechanisms producing time course of cerebellar long-term depression

Cerebellar long-term depression (LTD) is induced by short-lasting synaptic activities, progressively expressed, and then maintained for hours or longer. Short-lasting events, such as calcium transients, are activated and required for the induction of LTD. Further, a positive-feedback kinase loop was shown to follow the transient events and to aid the transition between LTD induction and prolonged synaptic depression. Yet, it is not entirely clear as to how LTD is maintained and how the maintenance mechanisms are activated, mainly because of a lack of experimental studies regarding this topic, while an idea has been theoretically proposed. A new analysis of the experimental results suggests that early maintenance mechanisms display a threshold behavior and that they may be of stochastic nature. This suggestion is conceptually consistent with an idea from a computational study, which postulates that other bistable switch systems are required for LTD maintenance. We thus propose that cellular mechanisms showing a threshold behavior and a stochastic nature maintain LTD, and that future experimental studies in search of such mechanisms would be an important step toward fully understanding the time course of LTD.

Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins.

Timely regulated sorting from early to late endosomes is required to maintain cerebellar long-term depression

An important feature of long-term synaptic plasticity is the prolonged maintenance of plastic changes in synaptic transmission. The trafficking of AMPA-type glutamate receptors (AMPARs) is involved in the expression of many forms of synaptic plasticity, yet the subsequent events accomplishing the maintenance of plastic changes in synaptic AMPAR numbers are not fully understood. Here, we find that maintenance of cerebellar long-term depression results from a reduction in the number of AMPARs residing within endocytic recycling pathways. We then develop a genetically encoded, photosensitive inhibitor of late endosome sorting and use this to discover that initial maintenance of long-term depression relies on timely regulated late endosome sorting, which exhibits a threshold as well as switch-like behavior. Thus, our results indicate that recycling AMPAR numbers are reduced by a switching machinery of transient late endosome sorting, and that this process enables the transition from basal synaptic transmission to long-term depression maintenance.Long term depression (LTD) of the cerebellum is known to be mediated by postsynaptic trafficking of glutamate receptor AMPAR. Here, Kim and colleagues show that early- to late-endosomal sorting of AMPAR represents the switch from expression to maintenance phase of cerebellar LTD.

A Model of Induction of Cerebellar Long-Term Depression Including RKIP Inactivation of Raf and MEK

We report an updated stochastic model of cerebellar Long Term Depression (LTD) with improved realism. Firstly, we verify experimentally that dissociation of Raf kinase inhibitor protein (RKIP) from Mitogen-activated protein kinase kinase (MEK) is required for cerebellar LTD and add this interaction to an earlier published model, along with the known requirement of dissociation of RKIP from Raf kinase. We update Ca2+ dynamics as a constant-rate influx, which captures experimental input profiles accurately. We improve α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptor interactions by adding phosphorylation and dephosphorylation of AMPA receptors when bound to glutamate receptor interacting protein (GRIP). The updated model is tuned to reproduce experimental Ca2+ peak vs. LTD amplitude curves at four different Ca2+ pulse durations as closely as possible. We find that the updated model is generally more robust with these changes, yet we still observe some sensitivity of LTD induction to copy number of the key signaling molecule Protein kinase C (PKC). We predict natural variability in this number by stochastic diffusion may influence the probabilistic LTD response to Ca2+ input in Purkinje cell spines and propose this as an extra source of stochasticity that may be important also in other signaling systems.

The cytosolic Ca2+ concentration in acutely dissociated subfornical organ (SFO) neurons of rats: Spontaneous Ca2+ oscillations and Ca2+ oscillations induced by picomolar concentrations of angiotensin II

Characteristics of subfornical organ (SFO) neurons were examined by measuring the cytosolic Ca2+ concentration ([Ca2+]i) in acutely dissociated neurons of the rat. SFO neurons, defined by the responsiveness to 50 mM K+ (n = 67) responded to glutamate (86%), angiotensin II (AII) (50%), arginine vasopressin (AVP) (66%) and/or carbachol (CCh) (61%), at their maximal concentrations, with marked increases in [Ca2+]i. More than a half (174/307) of SFO neurons examined exhibited spontaneous Ca2+ oscillations, while the remainder showed a relatively stable baseline under unstimulated conditions. Spontaneous Ca2+ oscillations were suppressed when extracellular Ca2+ was removed and were inhibited when extracellular Na+ was replaced with equimolar N-methyl-D-glucamine. Ca2+ oscillations were unaffected by the inhibitor of Ca2+-dependent ATPases cyclopiazonic acid, the N-type Ca2+ channel blocker ω-conotoxin GVIA and the P/Q-type Ca2+ channel blocker ω-agatoxin IVA, but significantly inhibited by the high-voltage-activated Ca2+ channel blocker Cd2+ and the L-type Ca2+ channel blocker nicardipine. Ca2+ oscillations were also completely arrested by the voltage-gated Na+ channel blocker tetrodotoxin in 50% of SFO neurons but only partially in the remaining neurons. These results suggest that SFO neurons exhibit spontaneous membrane Ca2+ oscillations that are dependent in part on Ca2+ entry through L-type Ca2+ channels, whose activation may result from burst firing. Moreover, AII at picomolar concentrations induced Ca2+ oscillations in neurons showing no spontaneous Ca2+ oscillations, while spontaneous Ca2+ oscillations were arrested by gamma-aminobutyric acid (10 μM), suggesting that rises in [Ca2+]i during Ca2+ oscillations may play an important role in the modulation of SFO neuron function.

Analysis of G-protein-activated inward rectifying K+ (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons

While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.