Cyclin-dependent kinase 5 is required for normal cerebellar development.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its kinase activity is dependent upon its association with either of the activating subunits p35 or p39, which are mainly expressed in neurons. We previously reported that Cdk5 knockout (KO) mice exhibit perinatal lethality, defective neuronal migration, and abnormal positioning of neurons in the facial motor nucleus and inferior olive in the hindbrain and Purkinje cells (PCs) in the cerebellum. In this study, we focused on the analysis of the role of Cdk5 in cerebellar development. For this purpose we generated midbrain-hindbrain-specific Cdk5 conditional knockout (MHB-Cdk5 KO) mice because the cerebellum develops postnatally, whereas Cdk5 KO mice die perinatally. Histological analysis of the MHB-Cdk5 KO mice revealed a significant size reduction of the cerebellum. In addition, profound disturbance of inward migration of granule cells (GC) was observed in the developing cerebellum. A normal dendritic development of the Purkinje cells (PCs) was disturbed in MHB-Cdk5 KO mice. Cultured Cdk5-null PCs showed similar dendritic abnormalities. These results indicate that Cdk5/p35 plays an important role in neuronal migration of PCs and GCs and dendrite formation of PCs in cerebellar development.

Dietary methionine level affects linoleic acid metabolism through phosphatidylethanolamine N-methylation in rats.

The effects of dietary methionine level on the profiles of fatty acids and phospholipids and on the plasma cholesterol concentration were investigated to confirm whether the methionine content of dietary proteins is one of the major factors that cause differential effects on lipid metabolism. The effect of dietary supplementation with eritadenine, which is shown to be a potent inhibitor of phosphatidylethanolamine (PE) N-methylation, was also investigated. Rats were fed six diets containing casein (100 g/kg) and amino acid mixture (86.4 g/kg) differing in methionine content (2.5, 4.5, and 7.5 g/kg) and without or with eritadenine supplementation (30 mg/kg) for 14 d. The ratio of arachidonic to linoleic acid of liver microsomal and plasma phosphatidylcholine (PC) was significantly increased as the methionine level of diet was elevated, indicating that dietary methionine stimulates the metabolism of linoleic acid. The PC/PE ratio of liver microsomes and the plasma cholesterol concentration were also increased by dietary methionine. These effects of methionine were completely abolished by eritadenine supplementation The S-adenosylmethionine concentration in the liver reflected the methionine level of diet. These results support the idea that the differential effects of dietary proteins on lipid metabolism might be ascribed, at least in part, to their different methionine contents, and that methionine might exert its effects through alteration of PE N-methylation.

Methionine content of dietary proteins affects the molecular species composition of plasma phosphatidylcholine in rats fed a cholesterol-free diet.

The effects of dietary protein types and methionine supplementation on phospholipid metabolism were investigated to clarify the mechanism of the hypocholesterolemic action of soybean protein in rats fed a cholesterol-free diet. The effect of switching from a casein diet to a soybean protein diet was also investigated. Rats were fed casein, soybean protein or soybean protein + methionine diet for 14 d. Compared with casein diet, feeding of soybean protein diet led to significantly higher proportions of linoleic acid and linoleic acid-containing molecular species, especially 16:0-18:2, in plasma and liver microsomal phosphatidylcholine (PC). In addition, significantly lower plasma cholesterol concentration, hepatic S-adenosylmethionine concentration and liver microsomal PC:phosphatidylethanolamine ratio resulted. These alterations caused by the soybean protein diet were significantly suppressed by supplementing methionine to the level of the casein diet (3.4 g/kg diet). The proportion of the sum of certain plasma PC molecular species, which contain 18:1 or 18:2 in the sn-2 position, increased in response to the switch from the casein diet to the soybean protein diet at a rate similar to the decrease in plasma cholesterol concentration; there was a significant correlation between the two variables (r = -0.992, P < 0.001). These results indicate that about 40% of the hypocholesterolemic action of soybean protein is due to the low methionine content of the protein and might be associated with alterations of the plasma phospholipid molecular species profile.

Behavioral study on the gracile axonal dystrophy (GAD) mutant mouse.

We investigated motor function and pain sensation in the gracile axonal dystrophy (GAD) mutant mouse, using the tail-flick test and the rotarod test. GAD (gad/gad) and normal sib mice (gad/+ or +/+) were used between 5 and 11 weeks of age, during which time the behavioral signs of GAD mice shifted from sensory ataxia (about 4 to 8 weeks of age) to paresis (after about 9 weeks of age). In the tail-flick test, significant shortening of latency was observed at 6 and 8 weeks of age in female GAD mice, in comparison with normal female mice. This may be related to dysfunction or degeneration of axons in the fasiculus gracilis, whose collaterals are thought to control the transmission of nociceptive information. In the rotarod test, a cumulative chi 2 test showed significant reduction in the performance times of GAD mice beginning at 5 and 6 weeks of age in males and females, respectively, indicating that the rotarod test can detect the development of motor incoordination as early as these ages. The performance times of GAD mice dropped sharply from 9 weeks of age onwards, and this is believed to reflect the progression of paresis. The rotarod test therefore appears to be a good method of quantifying behavioral changes in GAD mice and to be applicable both to objective selection of GAD mice before 8 weeks of age and to evaluation of drugs to treat ataxia or paresis.

Convulsions in senescence-accelerated mice (SAM-R/1/Eis).

Senescence-accelerated mice (SAM) are one of the animal models used for studying senescence, which consist of several substrains such as SAM-R/1, R/2, P/1, P/2. SAM-R/1/Eis maintained in Eisai Tsukuba Research Laboratories, Ibaraki, Japan, was originally introduced as a substrain of a normal control SAM-R/1 from Kyoto University, Japan. We have noted signs of convulsions in SAM-R/1/Eis mice during routine animal care, particularly while changing cages. We identified the clinical signs and determined the concentrations of glucose and immunoreactive insulin in plasma of SAM-R/1/Eis mice. There were no differences in the male:female ratios of mice showing prodrome only, grand mal, or no-signs. The ages at which prodrome and grand mal were first noted peaked between 20 and 25 weeks. Concentrations of glucose and immunoreactive insulin in plasma did not indicate the mice were in insulin hypoglycemia, which is one cause of convulsions. AKR strain mice, some of which originated with the SAM strain are known to become convulsive by repeated "throwing" stimulations. Conversely, in SAM-R/1/Eis, throwing stimuli are not needed to cause convulsive signs. Thus it is likely that in SAM-R/1/Eis mice the signs are triggered by repeating mild environmental changes, such as changing cages. The results of this study show that SAM-R/1/Eis is neither a normal control strain, nor an original SAM-R/1 strain. But it is possible that SAM-R/1/Eis is another useful animal model for studying spontaneous convulsion.