Faculty Bibliography

Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies

BACKGROUND: The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2-3 months) and old (10-14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8-9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15-17 h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n = 85), which did not differ from that (92%, n = 106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence

Sex steroids play important and diverse roles in the regulation of structure and function of the central nervous system. Early in life, steroids shape the structure of sensitive areas of the brain, especially those involved in the control of reproductive behavior and ovarian function. Original studies demonstrating organizing effects of steroids on the brain were carried out in rodents, but more recently these studies have been extended to primates, including humans. Throughout life, sex steroids regulate neural function by influencing steroid receptor-bearing neurons and by influencing neurons via steroid receptor-independent mechanisms. Sex steroid receptors have been identified in the brain, especially in the phylogenetically ancient structures that regulate reproductive behavior. Sex steroids that affect neural function can originate peripherally from the brain and/or adrenal gland, and can be synthesized within the brain itself. A number of neurally active progestogens and androgens are synthesized de novo in the brain, and estrogens can be converted within the brain from androgens by the enzyme aromatase. Thus, ovarian and central nervous system sex steroids play important roles in regulating reproductive behavior by regulating neural structure and function

To determine estrogen effects on osmotic regulation of arginine vasopressin (AVP) and body fluids, we suppressed endogenous estrogen and progesterone using the gonadotropin-releasing hormone (GnRH) analog leuprolide acetate (GnRHa). Subjects were assigned to one of two groups: 1) GnRHa alone, then GnRHa + estrogen (E, n = 9, 25 +/- 1 yr); 2) GnRHa alone, then GnRHa + estrogen with progesterone (E/P, n = 6, 26 +/- 3). During GnRHa alone and with hormone treatment, we compared AVP and body fluid regulatory responses to 3% NaCl infusion (HSI, 120 min, 0.1 ml. min(-1). kg body wt(-1)), drinking (30 min, 15 ml/kg body wt), and recovery (60 min of seated rest). Plasma [E(2)] increased from 23.9 to 275.3 pg/ml with hormone treatments. Plasma [P(4)] increased from 0.6 to 5.7 ng/ml during E/P and was unchanged (0.4 to 0.6 ng/ml) during E. Compared with GnRHa alone, E reduced osmotic AVP release threshold (275 +/- 4 to 271 +/- 4 mosmol/kg, P < 0.05), and E/P reduced the AVP increase in response during HSI (6.0 +/- 1.3 to 4.2 +/- 0.6 pg/ml at the end of HSI), but free water clearance was unaffected in either group. Relative to GnRHa, pre-HSI plasma renin activity (PRA) was greater during E (0.8 +/- 0.1 vs. 1.2 +/- 0.2 ng ANG I. ml(-1). h(-1)) but not after HSI or recovery. PRA was greater than GnRHa during E/P at baseline (1.1 +/- 0.2 vs. 2.5 +/- 0.6) and after HSI (0.6 +/- 0.1 vs. 1.1 +/- 1.1) and recovery (0.5 +/- 0.1 vs. 1.3 +/- 0.2 ng ANG I. ml(-1). h(-1)). Baseline fractional excretion of sodium was unaffected by E or E/P but was attenuated by the end of recovery for both E (3.3 +/- 0.6 vs. 2.4 +/- 0.4%) and E/P (2.8 +/- 0.4 vs 1.7 +/- 0.4%, GnRHa alone and with hormone treatment, respectively). Fluid retention increased with both hormone treatments. Renal sensitivity to AVP may be lower during E due to intrarenal effects on water and sodium excretion. E/P increased sodium retention and renin-angiotensin-aldosterone stimulation

The delivery of copper to mammary gland and milk and the effects of lactation were examined in rats. Traces of (67)Cu/(64)Cu(II) were injected intraperitoneally or intravenously into virgin rats or lactating rats (2-5 days postpartum), and incorporation into blood, milk, and tissues was monitored. In virgin rats, most of the isotope first entered the liver and kidney. In lactating rats, almost 60% went directly to the mammary gland. Uptake rates and copper contents of the mammary gland were 20-fold higher in lactation. (67)Cu/(64)Cu appeared in milk and milk ceruloplasmin as rapidly as in mammary tissue and when there was no (67)Cu/(64)Cu-ceruloplasmin in the maternal plasma. Plasma (125)I-labeled albumin entered milk much more slowly. Milk ceruloplasmin (10 mg/l) had 25% of the (67)Cu/(64)Cu. Milk copper was 3.3 mg/l. Thus lactation markedly enhances the avidity of the mammary gland for copper, diverting most of it from liver and kidney to that tissue. Also, the primary source of milk ceruloplasmin is the mammary gland and not the maternal plasma.

In murine L cells, treatment with calpeptin or calpain inhibitor III increased Abeta42, but not Abeta40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the beta-cleaved CTF showed that generation of both alpha- and beta-cleaved CTFs increase proportionately following inhibition of calpains. Pulse-chase metabolic labeling confirmed that inhibiting calpains increases the production of alpha- and beta-cleaved APP metabolites. Immunolabeling showed greater betaCTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/luminal epitope found on both APP and betaCTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both alpha- and beta-cleavage without changing the ratio of alphaCTFs/betaCTFs, and 3) the bulk of the betaCTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate 'receptor' domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119-144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced alpha-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains

Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction

We examined whether Gbx2 is required after embryonic day 9 (E9) to repress Otx2 in the cerebellar anlage and position the midbrain/hindbrain organizer. In contrast to Gbx2 null mutants, mice lacking Gbx2 in rhombomere 1 (r1) after E9 (Gbx2-CKO) are viable and develop a cerebellum. A Gbx2-independent pathway can repress Otx2 in r1 after E9. Mid/hindbrain organizer gene expression, however, continues to be dependent on Gbx2. We found that Fgf8 expression normally correlates with the isthmus where cells undergo low proliferation and that in Gbx2-CKO mutants this domain is expanded. We propose that Fgf8 permits lateral cerebellar development through repression of Otx2 and also suppresses medial cerebellar growth in Gbx2-CKO embryos. Our work has uncovered distinct requirements for Gbx2 during cerebellum formation and provided a model for how a transcription factor can play multiple roles during development

Mice deficient in interleukin (IL)-2 production or the IL-2 receptor alpha or beta chains develop a lethal autoimmune syndrome. CD4(+) regulatory T cells have been shown to prevent autoimmune diseases, allograft rejection, and to down-regulate antibody responses against foreign antigens. To assess the role of IL-2 in the generation and function of regulatory T cells, we transferred CD4(+) T cells from mice genetically deficient in IL-2 or IL-2R(alpha) (CD25) expression. A small number of splenic or thymic CD4(+) T cells from IL-2 knockout mice can protect mice from spontaneous experimental autoimmune encephalomyelitis (EAE). In contrast, splenic or thymic CD4(+) T cells from CD25 knockout donor mice conferred little or no protection. We conclude that T cells with regulatory potential can develop, undergo thymic selection, and migrate to the peripheral lymphoid organs in the absence of IL-2, and do not protect from disease by means of IL-2 secretion. However, IL-2 signaling in regulatory T cells is essential for their protective function. Altogether, our results favor a model whereby IL-2 induces regulatory T cell activity