Akira Futatsugi

Two types of ryanodine receptors in mouse brain: Skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons

Two types of ryanodine receptors, channels for Ca2+ release from intracellular stores, are known. We detected the skeletal muscle type only in cerebellum by immunoblot analysis of microsomes and partially purified proteins. The cardiac muscle type was found in all parts of the mouse brain. Immunohistochemical study showed that the cardiac muscle type was localized mainly at the somata of most neurons. Analysis of mutant cerebella suggested that the skeletal muscle type was present exclusively in Purkinje cells. These results suggest that Ca(2+)-induced Ca2+ release, probably mediated by the cardiac muscle receptor, functions generally in various neurons, whereas depolarization-induced Ca2+ release, probably mediated by the skeletal muscle receptor, functions specifically in Purkinje cells.

M3 muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice

The M1 and M3 subtypes are the major muscarinic acetylcholine receptors in the salivary gland and M3 is reported to be more abundant. However, despite initial reports of salivation abnormalities in M3‐knockout (M3KO) mice, it is still unclear which subtype is functionally relevant in physiological salivation. In the present study, salivary secretory function was examined using mice lacking specific subtype(s) of muscarinic receptor. The carbachol‐induced [Ca2+]i increase was markedly impaired in submandibular gland cells from M3KO mice and completely absent in those from M1/M3KO mice. This demonstrates that M3 and M1 play major and minor roles, respectively, in the cholinergically induced [Ca2+]i increase. Two‐dimensional Ca2+‐imaging analysis revealed the patchy distribution of M1 in submandibular gland acini, in contrast to the ubiquitous distribution of M3. In vivo administration of a high dose of pilocarpine (10 mg kg−1, s.c.) to M3KO mice caused salivation comparable to that in wild‐type mice, while no salivation was induced in M1/M3KO mice, indicating that salivation in M3KO mice is caused by an M1‐mediated [Ca2+]i increase. In contrast, a lower dose of pilocarpine (1 mg kg−1, s.c.) failed to induce salivation in M3KO mice, but induced abundant salivation in wild‐type mice, indicating that M3‐mediated salivation has a lower threshold than M1‐mediated salivation. In addition, M3KO mice, but not M1KO mice, had difficulty in eating dry food, as shown by frequent drinking during feeding, suggesting that salivation during eating is mediated by M3 and that M1 plays no practical role in it. These results show that the M3 subtype is essential for parasympathetic control of salivation and a reasonable target for the drug treatment and gene therapy of xerostomia, including Sjögrens syndrome.

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors (IP3R2 and IP3R3, respectively) from mouse lung and found a novel alternative splicing segment, SIm2, at 176–208 of IP3R2. The long form (IP3R2 SIm2+) was dominant, but the short form (IP3R2 SIm2–) was detected in all tissues examined. IP3R2 SIm2– has neither IP3 binding activity nor Ca2+ releasing activity. In addition to its reticular distribution, IP3R2 SIm2+ is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca2+ ionophore stimulation, most of the IP3R2 SIm2+ is in clusters. IP3R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca2+ ionophore stimulation. IP3R2 SIm2– does not form clusters in either resting or stimulated cells. IP3 binding-deficient site-directed mutants of IP3R2 SIm2+ and IP3R3 fail to form clusters, indicating that IP3 binding is involved in the cluster formation by these isoforms. Coexpression of IP3R2 SIm2– prevents stimulus-induced IP3R clustering, suggesting that IP3R2 SIm2– functions as a negative coordinator of stimulus-induced IP3R clustering. Expression of IP3R2 SIm2– in CHO-K1 cells significantly reduced ATP-induced Ca2+ entry, but not Ca2+ release, suggesting that the novel splice variant of IP3R2 specifically influences the dynamics of the sustained phase of Ca2+ signals.

Cell adhesion molecules regulate Ca2+-mediated steering of growth cones via cyclic AMP and ryanodine receptor type 3

Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89–93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909–916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.

Immunocytochemical localization of the α1A subunit of the P/Q-type calcium channel in the rat cerebellum

Among various types of low‐ and high‐threshold calcium channels, the high voltage‐activated P/Q‐type channel is the most abundant in the cerebellum. These P/Q‐type channels are involved in the regulation of neurotransmitter release and in the integration of dendritic inputs. We used an antibody specific for the α1A subunit of the P/Q‐type channel in quantitative pre‐embedding immunogold labelling combined with three‐dimensional reconstruction to reveal the subcellular distribution of pre‐ and postsynaptic P/Q‐type channels in the rat cerebellum. At the light microscopic level, immunoreactivity for the α1A protein was prevalent in the molecular layer, whereas immunostaining was moderate in the somata of Purkinje cells and weak in the granule cell layer. At the electron microscopic level, the most intense immunoreactivity for the α1A subunit was found in the presynaptic active zone of parallel fibre varicosities. The dendritic spines of Purkinje cells were also strongly labelled with the highest density of immunoparticles detected within 180 nm from the edge of the asymmetrical parallel fibre–Purkinje cell synapses. By contrast, the immunolabelling was sparse in climbing fibre varicosities and axon terminals of GABAergic cells, and weak and diffuse in dendritic shafts of Purkinje cells. The association of the α1A subunit with the glutamatergic parallel fibre–Purkinje cell synapses suggests that presynaptic channels have a major role in the mediation of excitatory neurotransmission, whereas postsynaptic channels are likely to be involved in depolarization‐induced generation of local calcium transients in Purkinje cells.

Tumor Necrosis Factor-α Regulates Cyclin-dependent Kinase 5 Activity during Pain Signaling through Transcriptional Activation of p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. We have previously reported that Cdk5 participates in the regulation of nociceptive signaling, and the expression of Cdk5 and its activator, p35, are up-regulated in nociceptive neurons during peripheral inflammation. The aim of our current study was to identify the proinflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that tumor necrosis factor-α (TNF-α) activates p35 promoter activity in a dose- and time-dependent manner and concomitantly up-regulates Cdk5 activity. Because TNF-α is known to activate ERK1/2, p38 MAPK, JNK, and NF-κB signaling pathways, we examined their involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK, and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of Egr-1, a transcription factor, were increased by TNF-α treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which carrageenan injection into the hind paw causes hypersensitivity to heat stimuli, TNF-α mRNA was increased at the site of injection. These findings suggest that TNF-α-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling.

Phosphorylation of Homer3 by Calcium/Calmodulin-Dependent Kinase II Regulates a Coupling State of Its Target Molecules in Purkinje Cells

Homer proteins are components of postsynaptic density (PSD) and play a crucial role in coupling diverse target molecules. However, the regulatory aspect of Homer-mediated coupling has been addressed only about a dominant-negative effect of Homer1a, which requires de novo gene expression. Here, we present evidence that Homer-mediated coupling is regulated by its phosphorylation state. We found that Homer3, the predominant isoform in Purkinje cells, is phosphorylated by calcium/calmodulin-dependent protein kinase II (CaMKII) both in vitro and in vivo. Biochemical fractionation with phosphor-specific antibodies revealed the presence of phosphorylated Homer3 in the cytosolic fraction in contrast to high levels of nonphosphorylated Homer3 in PSD. In P/Q-type voltage-gated-Ca2+ channel knock-out mice, in which CaMKII activation was reduced, the levels of Homer3 phosphorylation and the soluble form of Homer 3 were markedly lower. Furthermore, both robust phosphorylation of Homer3 and its dissociation from metabotropic glutamate receptor 1α (mGluR1α) were triggered by depolarization in primary cultured Purkinje cells, and these events were inhibited by CaMKII inhibitor. An in vitro binding kinetic analysis revealed that these phosphorylation-dependent events were attributable to a decrease in the affinity of phosphorylated Homer3 for its ligand. In a heterologous system, the Ca2+ signaling pattern induced by mGluR1α activation was modulated by the Homer3 phosphorylation state. Together, these findings suggested that Homer3 in Purkinje cells might function as a reversible coupler regulated by CaMKII phosphorylation and that the phosphorylation is capable of regulating the postsynaptic molecular architecture in response to synaptic activity.

Cyclin-dependent kinase 5 regulates E2F transcription factor through phosphorylation of Rb protein in neurons

Recent studies have shown the involvement of cyclin-dependent kinase 5 (Cdk5) in cell cycle regulation in postmitotic neurons. In this study, we demonstrate that Cdk5 and its co-activator p35 were detected in the nuclear fraction in neurons and Cdk5/p35 phosphorylated retinoblastoma (Rb) protein, a key protein controlling cell cycle re-entry. Cdk5/p35 phosphorylates Rb at the sites similar to those phosphorylated by Cdk4 and Cdk2. Furthermore, increased Cdk5 activity elevates activity of E2F transcription factor, which can trigger cell cycle re-entry, leading to neuronal cell death. A normal Cdk5 activity in neurons did not induce E2F activation, suggesting that Cdk5 does not induce cell cycle re-entry under normal conditions. Taken together, these results indicate that Cdk5 can regulate cell cycle by its ability to phosphorylate Rb. Most importantly, increased Cdk5 activity induces cell cycle re-entry, which is especially detrimental for survival of postmitotic neurons.

Knockdown of Cav2.1 calcium channels is sufficient to induce neurological disorders observed in natural occurring Cacna1a mutants in mice

The CACNA1A gene encodes the poreforming, voltage-sensitive subunit of the voltage-dependent Ca(v)2.1 calcium channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. In mice, mutations of the homolog Cacna1a cause recessively inherited phenotypes in tottering, rolling Nagoya, rocker, and leaner mice. Here we describe two knockdown mice with 28.4+/-3.4% and 13.8+/-3.3% of the wild-type Ca(v)2.1 quantity. 28.4+/-3.4% level mutants displayed ataxia, absence-like seizures and progressive cerebellar atrophy, although they had a normal life span. Mutants with 13.8+/-3.3% level exhibited ataxia severer than the 28.4+/-3.4% level mutants, absence-like seizures and additionally paroxysmal dyskinesia, and died premature around 3 weeks of age. These results indicate that knock down of Ca(v)2.1 quantity to 13.8+/-3.3% of the wild-type level are sufficient to induce the all neurological disorders observed in natural occurring Cacna1a mutants. These knockdown animals with Ca(v)2.1 calcium channels intact can contribute to functional studies of the molecule in the disease.

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5‐trisphosphate (IP3) receptors (IP3R2 and IP3R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5‐Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild‐type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP3R2 and IP3R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP3R2s and IP3R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.