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.

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.

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 A TTR. 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.

A brp homolog of Haloarcula japonica: gene cloning and transcriptional analysis.

The gene encoding a homolog of Halobacterium salinarum bacterioopsin-related protein (Brp) was cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible brp gene revealed that the structural gene consisted of an open reading frame of 1,062 nucleotides encoding 354 amino acids. Transcription of the brp homolog in Ha. japonica was confirmed by RT-PCR.