Eiki Takahashi

Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice

BackgroundADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22s sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice.ResultsADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed.ConclusionThe results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy.

Chronic Intracerebroventricular Administration of Relaxin-3 Increases Body Weight in Rats

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.

Deficits in spatial learning and motor coordination in ADAM11-deficient mice

BackgroundADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting.ResultsADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice.ConclusionOur results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes.

Altered nociceptive response in ADAM11-deficient mice.

Mice that lack A Disintegrin And Metalloprotease 11 (ADAM11) protein showed normal responses to stimuli in the von Frey test and the hot plate test, but showed reduced responses in the formalin paw test and acetic acid writhing test. Our results indicate that the cell adhesion-related molecule ADAM11 may play a role in pain transmission and in inflammatory regulation mechanisms underlying changes in the threshold for pain perception.

Expression analysis of P/Q-type Ca2+ channel α1A subunit mRNA in olfactory mitral cell in N-type Ca2+ channel α1B subunit gene-deficient mice

Abstract N-type and P/Q-type Ca 2+ channels play an important role in the processing of olfactory information. However, N-type Ca 2+ channel α 1B -deficient mice show normal behavior, presumably owing to compensation by other Ca 2+ channels. P/Q-type Ca 2+ channel α 1A mRNA was expressed at a higher level in olfactory bulb of homozygous α 1B -deficient mice than wild-type or heterozygous mice. LacZ expression in olfactory mitral cells of homozygous α 1B -deficient x α 1A 1.5-lacZ mice, carrying a 1.5-kb 5′-upstream fragment of the α 1A gene fused to the lacZ reporter gene, was increased compared to that in wild-type or heterozygous mice. Therefore, a possible explanation for the normal behavior of α 1B -deficient mice is compensation by the α 1A gene and that the 1.5-kb 5′-upstream region of this gene contains an enhancer cis -element for compensation in olfactory mitral cells.

Neuron-specific expression of reporter gene in transgenic mice carrying the 5'-upstream region of mouse P/Q-type Ca2+ channel α1A subunit gene fused to E. coli lacZ reporter gene

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel alpha 1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5-upstream region failed to show reporter expression. Histological examinations showed that the three 5-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5-upstream region. These results suggest that the expression of the mouse P/Q-type Ca2+ channel alpha 1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.

Expression analysis of Escherichia coli lacZ reporter gene in transgenic mice.

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of beta-galactosidase activity.

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca2+ channel α1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, α1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel α1 or β subunit. In this study, we studied the levels of α1A, α1C, α1D, α1E, β1, β2, β3 and β4 mRNAs in adrenal gland of α1B-deficient mice. The α1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of α1C, α1D, α1E, β1, β2, β3 and β4 was found among wild, heterozygous and homozygous mice. The protein level of α1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5′-upstream region of α1A gene, we examined lacZ expression in α1B-deficient × α1A6.3-lacZ mice (carrying a 6.3-kb 5′-upstream fragment of α1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous α1B-deficient × α1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of α1B-deficient mice is that compensation by α1A subunit occurs and that 6.3-kb 5′-upstream region of α1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)

Genetic Background Influences P/Q-type Ca2+ Channel α1A Subunit mRNA Expression in Olfactory Bulb and Reproductive Ability of N-type Ca2+ Channel α1B Subunit-Deficient Mice

The Ca2+ channel α1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although the N-type Ca2+ channel plays a role in a variety of neuronal functions, α1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca2+ channel α1A subunit gene in olfactory bulb of α1B-deficient mice with a CBA × C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the α1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous α1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences α1A subunit mRNA expression and reproductive ability in α1B-deficient mice.

Expression analysis of the 5′-upstream region of mouse P/Q-type Ca2+ channel α lA subunit gene fused to Escherichia coli lacZ reporter gene in the spinal cord using transgenic mice

The P/Q-type Ca(2+) channel alpha(1A) subunit is expressed in spinal cord including ventral motor neurons and interneurons and dorsal horn. To identify the transcriptional mechanisms of the mouse alpha(IA) subunit gene in spinal cord, transgenic mice carrying a 0.5, 1.5, 3.0 or 6.3-kb 5-upstream region fused to the Escherichia coli lacZ reporter gene were examined. Transgenic mice carrying the 3.0-kb region expressed the reporter gene in dorsal horn and interneurons of ventral horn, although those with the 0.5-kb, 1.5-kb or 6.3-kb region did not. No transgenic mice expressed the reporter gene in motor neurons of ventral horn. These results suggest that in spinal cord, the expression mechanisms of the alpha(1A) subunit gene are complex, involving both positive and negative cis-regulatory elements, and the 6.3-kb 5-upstream region alone is not sufficient for the expression.