The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP 3 R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)-PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP 3 R1 specifically in PCs (L7-Cre;Itpr1 flox/flox ) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF-PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1 flox/flox mice. Our findings reveal a specific role for IP 3 R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF-PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.

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BACKGROUND: Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca 2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca 2+ /CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca 2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca 2+ /CaM/ CaMKKβ signaling through regulatory phosphorylation.

Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIβ promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIβ, which results in dissociation of the CaMKIIβ/ F-actin complex. Defective function of the PKC-mediated CaMKIIβ phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP 3 R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIβ through the mGluR/IP 3 R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells.n the cerebellum, Purkinje cells are the sole output from the neural circuit of the cerebellar cortex, and integrate numerous synaptic inputs (1). Spines along the distal dendrites of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites of cerebellar synaptic plasticity (1, 2). Spine density and morphology of Purkinje cells change significantly during development (3, 4), and morphological abnormalities of spines are closely associated with many neurological disorders (5-7). Recent studies also demonstrated that some forms of training for cerebellar motor learning results in altered spine density in Purkinje cells (8,9). Thus, maintenance of proper spine density and morphology of Purkinje cells is a critical aspect of cerebellar functions. However, the precise molecular mechanisms that maintain Purkinje cell spine density and morphology remain unclear.The actin filaments are a major structural element of the regulation of dendritic spine formation and morphology of neurons (10, 11). The actin dynamics in spines are regulated by many actinrelated molecules including Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) (12), which is one of the most abundant proteins in the brain (13). Among CaMKII isoforms (α, β, γ, and δ), CaMKIIβ possesses a specific F-actin binding domain (14) and plays an important role for regulating dendritic spine structure in hippocampal neurons. It is reported that suppression of CaMKIIβ expression leads to reduced spine formation, and conversely, overexpression of CaMKIIβ increases synapse number and motility of filopodia in hippocampal neurons (15,16). The effect of CaMKIIβ on maintaining mature spine structure requires its F-actin binding and bundling activity, but not its kinase activity (17). In addition, a recent study reported that in hippocampal neurons, activation of CaMKIIβ by postsynaptic Ca 2+ influx through the NMDA receptor and resultant autophosphorylation within the F-actin binding dom...