Sadahiro Ito

Mice lacking the glutamate-cysteine ligase modifier subunit are susceptible to myocardial ischaemia–reperfusion injury

AIMS Glutamate-cysteine ligase (GCL), a rate-limiting enzyme for glutathione (GSH) synthesis, is composed of catalytic and modifier subunits. This study examined the pathogenic role of GCL modifier subunits (GCLM) in myocardial ischaemia-reperfusion (I/R) injury using mice lacking the GCLM (GCLM(-/-)). METHODS AND RESULTS The GCLM(-/-)mice had an increase in myocardial I/R injury and apoptosis in ischaemic myocardium compared with GCLM(+/+) mice. There was a decrease in mitochondrial glutathione (GSH) levels in ischaemic myocardium that was more pronounced in GCLM(-/-) mice than in GCLM(+/+) mice (12 vs. 55% of baseline GCLM(+/+), respectively). The ESR signal intensity of the dimethyl-1-pyrroline-N-oxide-hydroxyl radical adducts in ischaemic myocardium was higher in GCLM(-/-) mice than in GCLM(+/+) mice. Hypoxia-reoxygenation induced greater mitochondrial damage in cultured cardiomyocytes from GCLM(-/-) mice than from GCLM(+/+) mice, as evidenced by a reduced membrane potential and increased protein carbonyl content in isolated mitochondria, together with enhanced cytochrome c translocation into the cytosol. Administration of GSH ethyl-ester attenuated myocardial I/R injury and reversed the mitochondrial damage in parallel with the mitochondrial GSH restoration in the myocardium or the cardiomyocytes of GCLM(-/-) mice. CONCLUSION GCLM(-/-) mice were susceptible to myocardial I/R injury partly through an increased vulnerability of mitochondria to oxidative damage owing to mitochondrial GSH reduction.

Avoiding the effect of linked genes is crucial to elucidate the role of Apcs in autoimmunity.

To the editor: It was reported that mice with targeted disruption of the gene that encodes serum amyloid P-component (Sap, encoded by Apcs) with a mixed genetic background (129/Sv × C57BL/6; F2) developed high titers of antibody to nuclear antigens (ANA) and severe glomerulonephritis, a phenotype resembling systemic lupus erythematosus (SLE)1. Thus, it was speculated that Sap contributes to the pathogenesis of SLE. But the Apcs gene on mouse chromosome 1 is located in a region harboring several genes reported to be associated with SLE susceptibility in multiple strain combinations2. This suggests that ignoring the confounding effect of genes around the disrupted Apcs gene in the knockout mice might lead to misinterpretation of the role of Sap in autoimmunity. To assess the effects of genetic background and genes linked to the disrupted Apcs gene, we first transferred the disrupted Apcs gene from Apcs–/– mice generated from 129/Sv//Evderived embryonic stem cells and backcrossed into C57BL/6 (129/Sv//Ev × C57BL/6; F4)3,4 to C3H/He mice as follows: a male Apcs–/– 129/ Sv//Ev × C57BL/6 mouse was mated with C3H/He female mice. Homozygous mutant (Apcs–/–) and wild-type (Apcs+/+) F2 mice from the heterozygote (Apcs+/–; F1) matings were analyzed. There was no significant difference in the titers of ANA between 12–13month-old Apcs–/– (n = 50; 27 females and 23 males) and Apcs+/+ (n = 41; 18 females and 23 males) 129/Sv//Ev × C57BL/6 × C3H/He littermates (Fig. 1a). The result indicates that Sap deficiency in itself does not promote autoimmunity. We then crossed the Apcs–/– 129/ Sv//Ev × C57BL/6 mice back into C57BL/6 for 10 generations to attain genetic homogeneity. The backcrossed 11–14-month-old (n = 37; 21 females and 16 males) Apcs–/– mice produced significantly higher titers of ANA than did Apcs+/+ (n = 29; 19 females, P = 0.00001, and 10 males, P = 0.0008) littermates (Fig. 1b). A linkage study indicated that the backcrossed Apcs–/–mice still carried at least a 6.2-cM 129/ Sv//Ev-type chromosome segment marked by D1Mit149, D1Mit206, D1Mit456 and Apcs, between D1Mit36 and D1Mit115. The segment overlaps the mouse SLE susceptibility loci Sle1 and Nba2 and harbors genes implicated in SLE susceptibility, such as Fcgr2b and interferonactivated gene 202 (Ifi202)2,5. Mice congenic for the Nba2 locus with a C57BL/6 genetic background (C57BL/6. Nba2) had increased levels of Ifi202 mRNA and protein in their spleen cells, which correlated with lupus susceptibility5. Similar to the Apcs–/– 129/Sv//Ev × C57BL/6 mice, the C57BL/6.Nba2 mice develop substantially higher titers of ANA relative to C57BL/6 mice but they never develop severe glomerulonephritis4,5. Thus, we compared the level of Ifi202 mRNA in the spleen of Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 littermates by northern blot hybridization. A 1.8-kilobase Ifi202 mRNA was detected in the Apcs–/– but not in the Apcs+/+ mice (Fig. 2a). Because the expression of Ifi202 is inducible by lipopolysaccharide (LPS), we compared the levels of Ifi202 mRNA in the spleen of Apcs–/–, Apcs+/– and Apcs+/+ mice at 4 h after intraperitoneal injection of Salmonella typhimirium LPS (15 mg/kg body weight). The levels of splenic Ifi202 mRNA of LPS-injected Apcs–/– and Apcs+/– mice were substantially higher than those of Apcs+/+ littermates and the Ifi202 mRNA level of Apcs–/– mice was approximately twice as high as that of Apcs+/– mice (Fig. 2a). Because transcription of Ifi202 is reportedly impaired in the C57BL/6 mouse5, the data suggest that the expression of the Ifi202 gene linked to the C57BL/6-derived wild-type Apcs gene is impaired but that linked to the 129/Sv//Ev-derived disrupted Apcs gene is not. The levels of splenic Ifi202 mRNA of wild-type 129/Sv//Ev and C3H/He mice are much the same and are substantially higher than those of wild-type C57BL/6 mice. Because the disrupted and wild-type Apcs alleles in the 129/Sv//Ev × C57BL/6 × C3H/He mice are derived from 129/Sv//Ev and C3H/He mice, respectively, the level of expression of the Apcs-linked Ifi202 gene between Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 × C3H/He mice is expected to be much the same. Indeed, no differences were detected in the Ifi202 mRNA levels between the Apcs–/– and Apcs+/+ mice as shown in Fig. 2b. Thus, differences in mean levels of ANA between the Apcs–/– and Apcs+/+ mice correlate with differences in the levels of splenic Ifi202 mRNA independent of Sap: no difference in the titers of ANA between the Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 × C3H/He mice was detected, whereas the Apcs–/– 129/Sv//Ev × C57BL/6 mice produced significantly higher titers of ANA than control Apcs+/+ littermates (Fig. 1a,b). A N A ti te r 2,560 2,560

Deposition of transthyretin amyloid is not accelerated by the same amyloid in vivo

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported in several types of amyloidoses. Families with a variant transthyretin (TTR V30M)-associated familial amyloidotic polyneuropathy (FAP) exhibit genetic anticipation, with TTR V30M-amyloid depositing at an earlier age in successive generations. The molecular bases of anticipation in FAP have remained to be determined. We asked if administration of TTR-amyloid fibrils (ATTR) extracted from the heart of an FAP TTR V30M patient would accelerate ATTR deposition in transgenic mice expressing the human mutant ttr gene responsible for FAP TTR V30M and indeed the administration did accelerate deposition of apolipoprotein A-II- amyloid fibrils (AApoAII), and not ATTR. Our experiments present, for the first time, evidence that the degree of inducibility of ATTR is low relative to AApoAII and we suggest that administration of ATTR may not explain the genetic anticipation which occurs in FAP.

Homozygous serum amyloid P component= deficiency does not enhance regression of AA amyloid deposits

Serum amyloid P component (SAP) is a common protein constituent of all types of amyloid deposits. Using SAP-deficient mice generated through gene targeting, we and others have shown that SAP significantly promotes amyloid deposition. It has been speculated that SAP protects amyloid fibrils from degradation by coating their exterior surface. To assess potential ways of treating individuals with amyloidosis, we examined the persistence of splenic AA amyloid fibrils in SAP-deficient and wild-type mice. No enhancement in the rate of regression of splenic AA amyloid was observed in the SAP-deficient mice relative to wild-type mice. These results present, for the first time, evidence that lack of SAP in AA amyloid deposits does not enhance regression of the deposits in vivo and suggest that dissociation of bound SAP from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo.

Mouse Models of Transthyretin Amyloidosis

Transthyretin (TTR) amyloidosis is caused by mutations in the TTR gene. Several observations, however, suggest the presence of factors, other than a mutation in the TTR gene, which affect TTR amyloid deposition. Although liver transplantation is the only curative treatment for TTR amyloidosis, its donor pool faces shortage, and TTR amyloid deposition continues in many patients after transplantation. Thus, some effective therapeutic strategies other than liver transplantation need to be developed. Mouse models of TTR amyloidosis would facilitate defining factors that accelerate amyloid deposition and would aid in developing effective treatments. Here, we summarize studies of transgenic mouse models of TTR amyloidosis in which questions were addressed about the role of various risk factors in the molecular pathogenesis of this intractable disease.

A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice

Src knockout mice show no detectable abnormalities in central nervous system (CNS) post-mitotic neurons, likely reflecting functional compensation by other Src family kinases. Cdk1- or Cdk5-dependent Ser75 phosphorylation in the amino-terminal Unique domain of Src, which shares no homology with other Src family kinases, regulates the stability of active Src. To clarify the roles of Src Ser75 phosphorylation in CNS neurons, we established two types of mutant mice with mutations in Src: phospho-mimicking Ser75Asp (SD) and non-phosphorylatable Ser75Ala (SA). In ageing SD/SD mice, retinal ganglion cell (RGC) number in whole retinas was significantly lower than that in young SD/SD mice in the absence of inflammation and elevated intraocular pressure, resembling the pathogenesis of progressive optic neuropathy. By contrast, SA/SA mice and wild-type (WT) mice exhibited no age-related RGC loss. The age-related retinal RGC number reduction was greater in the peripheral rather than the mid-peripheral region of the retina in SD/SD mice. Furthermore, Rho-associated kinase activity in whole retinas of ageing SD/SD mice was significantly higher than that in young SD/SD mice. These results suggest that Src regulates RGC survival during ageing in a manner that depends on Ser75 phosphorylation.