Koji Tomobe

Neurochemistry, neuropathology, and heredity in SAMP8: a mouse model of senescence.

The SAMP8 strain spontaneously develops learning and memory deficits with characteristics of aging, and is a good model for studying the mechanism of cognitive dysfunction with age. Oxidative stress occurs systemically in SAMP8 from early on in life and increases with aging. Neuropathological changes such as the deposition of Aβ, hyperphosphorylation of tau, impaired development of dendritic spines, and sponge formation, and neurochemical changes were found in the SAMP8 brain. These changes may be partially mediated by oxidative stress. Oxidative damage is a major factor in neurodegenerative disorders and aging. A decline in the respiratory control ratio suggesting mitochondrial dysfunction was found in the brain of SAMP8. The rise in oxidative stress following mitochondrial dysfunction may trigger neuropathological and neurochemical changes, disrupting the development of neural networks in the brain in SAMP8.

Impairment of CREB phosphorylation in the hippocampal CA1 region of the senescence-accelerated mouse (SAM) P8.

Senescence-accelerated mouse P8 (SAMP8) mice show deficits of learning and memory at an early age. However, no evidence of neurochemical changes was found in the hippocampus of SAMP8 at an early age. After electric shock in the passive avoidance test, SAMR1 (normal aging mice) showed biphasic responses in the phosphorylated CREB (p-CREB) level in the hippocampal CA1 region: an early peak detected at 1 to 3 h was followed by a marked drop at 6 h, and a second peak rise starting after 9 to 12 h after electric stimulation. On the other hand, SAMP8 manifested one peak in the p-CREB level 9 h after the stimulation. Since the phosphorylation of CREB plays an important role for synaptic plasticity and consolidation of long-term memory, the impairment of CREB phosphorylation in the hippocampal CA1 region of SAMP8 may cause learning and memory deficits.

Age-related changes of forkhead transcription factor FOXO1 in the liver of senescence-accelerated mouse SAMP8.

SAMP8 exhibits accelerated aging and a short lifespan. Insulin-like growth factor-1 receptor (IGF-1R)/FOXO pathway is associated with aging. Phosphorylation of IGF-1R, Akt, and FOXO1 was found to be increased during aging in the liver of SAMR1 normal aging mice. However, significant decreases in the phosphorylation of IGF-1R and Akt were observed in the liver of SAMP8 during aging compared with that in SAMR1, whereas phosphorylation of FOXO1 was markedly increased with age in SAMP8. In addition, the protein level of FOXO1 was decreased with age in SAMP8. Protein phosphatase 2A (PP2A) directly dephosphorylates FOXO1. Significant reduction of PP2A activity was observed in the liver nucleus of SAMP8. These results suggest the possibility that the increased FOXO1 phosphorylation might occur by the decreased activity of PP2A, resulting in the decrease in the protein level of FOXO1 in SAMP8. Furthermore, FOXO1 regulates longevity and the expression of antioxidant enzymes such as Mn-SOD and catalase. The expression of Mn-SOD and catalase was significantly decreased in the liver of SAMP8. Therefore, it is possible that the elevation of phosphorylated FOXO1 level with age causes a short lifespan in SAMP8.

Simultaneous determination for oxicam non-steroidal anti-inflammatory drugs in human serum by liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique was developed for the simultaneous determination of five non-steroidal anti-inflammatory oxicam drugs (ampiroxicam, tenoxicam, piroxicam, meloxicam and lornoxicam) in human plasma. These five oxicam drugs and isoxicam (internal standard) were extracted from human plasma with an Oasis(®) MAX cartridge column and analysed on a Unison UK-C18 column (2.0 mm × 100 mm, 3 μm) with an acetonitrile:10mM formic ammonium buffer (pH 3.0) (50:50) mobile phase at 0.20 ml/min at 37°C. The analytes were detected using a tandem mass spectrometer, equipped with an electrospray ion source (ESI). The instrument was used in multiple-reaction-monitoring (MRM) mode. The extraction yields from a 200 μl human plasma sample (containing 10 ng of each drugs) with the Oasis(®) MAX cartridge column were 93.3-102.5%. The detection limits were 0.01-6.5 ng/ml (S/N=3). Our developed method is very useful for the simultaneous determination of five oxicam (non-steroidal anti-inflammatory) drugs in human plasma by LC/MS/MS.

Localization of sialidase-positive cells expressing Mac-1 and immunoglobulin in the mouse thymus

We have sought an endogenous membrane bound sialidase acting at neutral pH in immune system, because the removal of sialic acid from cell surfaces will affect the cell-cell interaction directly or indirectly. The levels of activity of unique membrane-bound sialidase at neutral pH and also soluble sialidase are high in the thymus but low in the spleen and lymph nodes. These are thought to be plasma membrane and cytosolic types based on the behavior of inhibition by Cu2+ and 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid. Newly synthesized 5-bromo-4-chloro-3-indolyl-N-acetylnueraminic acid was used for histochemical staining of sialidase-positive thymic cells, and the results showed positive cells sparsely distributed in the corticomedullar region or medullary region of the thymus. They expressed immunoglobulin and Mac-1 antigen on their surfaces. These cells must therefore be of a B cell lineage, not a T cell lineage. We also found that some vessels in the thymus were sialidase-positive. Published in 2004.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of nine local anesthetic drugs.

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) procedure for the simultaneous determination of nine local anesthetic drugs (procaine, mepivacaine, lidocaine, ropivacaine, oxybuprocaine, tetracaine, bupivacaine, T-caine and dibucaine) in human serum is described. The chromatographic separation was performed on a Mightysil-RP-18 GP II column (2.0mm×150mm, particle size 5μm). The mobile phase consisted of 10mM acetic ammonium buffer (pH 5.4) and acetonitrile and was delivered at a flow rate of 0.20mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the nine local anesthetic drugs added to the human serum was performed with an Oasis(®) HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 10-100ng/mL. The recoveries of these drugs were in the range of 81.4-144%. The standard deviation (SD) values for all analytes were <0.10 for both intraday and interday accuracy and precision. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic applications. The sensitive and selective method offers the opportunity for the simultaneous screening and quantification, for clinical and forensic purposes, of almost all local anesthetics available in Japan.

Genetic analysis of learning and memory deficits in senescence-accelerated mouse (SAM)

Genetic analysis of learning and memory deficits (LMD) in senescence-accelerated mouse P8 (SAMP8) was performed by cross-mating SAMP8 and Japanese Fancy Mouse 1 (JF1). The incidence of LMD in the F2 generation showed a 3:1 segregation ratio of mice with LMD to normal mice, and the incidence of LMD in the backcross generation of the F1 to JF1 parental strain was in agreement with a 1:1 ratio of mice with LMD to normal mice. Estimation of the number of genes involved in the development of LMD using Wrights formula showed that at least two to four genes are involved. These results suggest that the inheritance of LMD is polygenically controlled and that there may be a single major gene, but this locus is not sex-linked. Moreover, hormonal influence on the development of LMD in SAMP8 females is of a genotype-dependent manner.

Quantitative trait loci for age-related memory dysfunction in SAMP8 and JF1 mice

Abstract The senescence-accelerated mouse (SAM) P8 strain exhibits severe age-related learning and memory deficits (LMD) well before the median age of survival. As a first step to clarify the genes involved in this deterioration, we have performed genetic analysis of SAMP8 using the whole genome scan for quantitative trait loci (QTLs) specifying the impairment in step-through passive avoidance response with F2 intercrosses of SAMP8 exhibiting short retention time (RT) and Japanese Fancy Mouse 1 (JF1) derived from Japanese wild mouse ( Mus musculus molossinus ) exhibiting normal long retention time. Genetic markers were typed at 113 loci spanning all chromosomes except the Y. Five loci have been identified with significant linkage to chromosomes 1, 12, 13 and 15 by interval mapping of 264 F2 mice. Three of them on chromosomes 1, 12, and 13 are due to SAMP8 background, while two of them on chromosome 15 are derived from JF1 background despite parental JF1 strain shows normal phenotype.

Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice.

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.

Genetic study of learning and memory deficits in SAMP8 mice

Abstract Cross-mating between SAMP8 and JF1 (Japanese Fancy Mouse 1) normal mice was performed for the genetic studies. At 5 months of age, learning and memory deficits (LMD) of F2 and backcross progenies were assessed using a Step-Through passive avoidance apparatus. The F2 generation showed a 3:1 segregation ratio of LMD to normal mice, and the backcross generation segregated into a 1:1 ratio of LMD to normal mice. These results are in good agreement with Mendelian inheritance. Estimation of the number of genes using Wrights formula showed that at least two to three genes are involved in the development of LMD in SAMP8 mice. Thus, LMD in SAMP8 mice may be inherited by a relatively small number of genes and regulated by a major single gene. In further experiments, we conducted QTL analysis to identify memory dysfunction QTLs in SAMP8 mice using 264 F2 progenies. A total of 126 genetic markers were typed in all chromosomes. Four significant QTLs to LMD were identified on chromosomes 1, 12, 13, and 15. The number of these QTLs is almost consistent with the statistically estimated gene number. Congenic strains carrying the QTLs need to be produced to verify the existence of LMD genes in the candidate regions of the chromosomes.