Shizu Hayasaka

Local Calcium Release in Dendritic Spines Required for Long-Term Synaptic Depression

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.

The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice ☆

The absence of smooth endoplasmic reticulum (SER) in the dendritic spine of Purkinje cells was found in dilute-lethal (dl) mouse cerebella as detected by immunohistochemistry using anti-inositol 1,4,5-triphosphate receptor antibody and electron microscopy. Since SER in the spine has been suggested to play a crucial role for synaptic regulation as an intracellular Ca2+ store (for reviews, see [Miller, R.J., Prog. Neurobiol., 37 (1991) 255-285: Simpson, P.B., Challiss, R.A.J. and Nahorski, S.R., Trends Neurosci., 18 (1995) 299-306]), a neurological defect, characterized by clonic convulsions with opisthotonus and ataxia, in the dilute-lethal mouse with homozygous trait may be attributable to the absence of SER in the dendritic spine of Purkinje cells.

Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat.

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.

Role of microglia in the pathogenesis of osmotic-induced demyelination.

Osmotic demyelination is a serious disease caused by rapid correction of hyponatremia. In humans, demyelinative lesions occur preferentially in the central pons, and thus are termed central pontine myelinolysis. Although accumulation of microglia has been reported in such demyelinative lesions, their role in the pathogenesis of osmotic demyelination remains unclear. We examined the expression of cytokines in microglia that accumulated in the demyelinative lesions in a rat model of osmotic demyelination. Hyponatremia was induced in rats by a combination of dDAVP infusion and liquid diet feeding. After 7 days, serum sodium levels were rapidly corrected by hypertonic saline injection. The rats developed severe motor deficits, and marked demyelinative lesions were found in the midbrain and cerebral cortex. In the area of the demyelinative lesions, massive accumulations of microglia were observed that expressed the proinflammatory cytokines TNF-alpha and IFN-gamma as well as iNOS. In contrast, in hyponatremia corrected rats treated with lovastatin, which is known to inhibit microglial infiltration in various animal models of CNS disease, neurological impairments and the degree of demyelination were significantly ameliorated. Lovastatin also reduced the accumulation of microglia and decreased the expression of TNF-alpha in the demyelinative lesions. These results indicate that microglia play a detrimental role in the pathogenesis of osmotic demyelination by producing proinflammatory cytokines, and further suggest that lovastatin may be useful in repressing the demyelination.

Increased expression of aquaporin-3 in the epidermis of DHCR24 knockout mice.

Background  The DHCR24 (3β‐hydroxysterol‐Δ24 reductase) gene encodes an enzyme catalysing conversion of desmosterol to cholesterol. Desmosterolosis is an autosomal recessive disease due to mutation in the DHCR24 gene, with low cholesterol and high desmosterol levels. To understand the pathophysiology of this disease, we utilized DHCR24 knockout mice and reported that DHCR24−/− mice die soon after birth. Their skin was less wrinkled, shiny, and revealed features of lethal restrictive dermopathy associated with severe defects in epidermal maturation and barrier function.

Α-Lipoic acid reduces congenital malformations in the offspring of diabetic mice

The mechanism of diabetes‐induced congenital malformation remains to be elucidated. It has been reported that α‐lipoic acid (LA) prevents neural tube defects (NTDs) in offsprings of rats with streptozotocin‐induced diabetes. Here, we evaluate the protective effect of LA against diabetic embryopathy, including NTDs, cardiovascular malformations (CVMs), and skeletal malformations, in mice.

Immunocytochemical detection and spatial distribution of myosin light‐chain kinase in preimplantation mouse embryos

As a follow-up to our previous study on the role of myosin light-chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme, in the development of preimplantation mouse embryos, we examined the presence and pattern of distribution of MLCK during preimplantation development of the mouse by whole-mount, indirect immunocytochemistry and by Western blotting, using a monoclonal antibody against MLCK. At all stages of preimplantation development, the nucleus was brightly stained with an unstained region around the nucleus, and regions near the cell membrane were also brightly stained. Using the optical sectioning capability of the confocal laser scanning microscope, we found that, up to the eight-cell stage, the regions of cell contact were mostly unstained, but along with the process of compaction, cell contact regions showed a clear staining pattern along with clearing of the cytoplasm. During formation of the blastocyst, a ring of immunofluorescence was found at the margin of the blastocoel. In the blastocyst, cells of the inner cell mass were less immunofluorescent than trophectoderm cells. These staining results appear to be due to specific immunoreaction between MLCK and the antibody, because the staining patterns were abolished when the antibody was preabsorbed by MLCK purified from chicken gizzard smooth muscle. In Western blotting of blastocysts, we found a band at 130 kD. We also show by immunoblotting and immunohistochemistry of various mouse tissues that the antibody used in this study has cross-reactivity to MLCK of various muscle and non-muscle tissues of the mouse. The presence and spatial distribution of MLCK at various stages of preimplantation development of the mouse suggest that it could play a crucial role in the regulation of the contractile events involved in the initial differentiation that occurs during formation of the mouse blastocyst.