Advancing age in human and experimental animals is associated with a profound change in the synthesis and secretion of steroid hormones. It appears that the cause of this problem is the inability of adrenocortical cells or Leydig cells of the aging rat model to effectively transport cholesterol to the cell's inner mitochondrial membrrane where the initial steps in steroid hormone production take place. Although, the various cellular and molecular mechanisms controlling this aging defect have not been definitely identified, considerable evidence from this laboratory points to excessive free radical formation and oxidative damage (especially from life-long continued processing of cholesterol for steroid production) to the cell machinery regulating the functional expression of crucial proteins, StAR, PBR/TSPO and potentially other StAR-related (StarD) proteins involved in cholesterol transport to inner mitochondrial membrane. The studies outlined in this proposal will explore these issues further and will specifically test the hypothesis that increased ROS formation and ensuing oxidative damage leads to changes in expression of sterol transfer proteins, StAR, and PBR/TSPO proteins in steroidogenic tissues model of aging animals and genetic mouse models of increased oxidative stress, and downstream, this leads to the transfer of less cholesterol to the inner mitochondrial membrane sites where cholesterol side chain cleavage takes place, and the first steroid, pregnenolone is formed. We also test a second hypothesis that pharmacological intervention with the use of small molecular weight synthetic antioxidants that reduce the degree of oxidative damage will prevent, reverse, or attenuate the age-related and oxidative stress dependent loss of steroidogenic response. To address these hypotheses, three Specific Aims are proposed.
Aim 1 is to establish Senescence-Accelerated-Mouse-Prone 8 (SAMP8) mice as a new aging model of impaired steroidogenesis. Using adrenal (or adrenocortical cells) and testicular Leydig cells from aging SAMP8 mice (and control SAMR1 mice) we will measure: a) hormonal regulation of steroidogensis, utilization of cholesterol from intracellular stores, hormone-induced cholesterol transport to mitochondria and expression of StAR and PBR/TSPO;b) cellular antioxidant levels, oxidative damage and antioxidant status;and c) alteration in the ASK1- p38 MAPK signaling cascade.
Aim 2 is to establish the causal role of oxidative stress in age-related decline in adrenal and testicular steroid hormone production. The first set of studies to be conducted will include monitoring changes in in vitro steroidogenesis, mRNA expression of StAR, PBR/TSPO and StarD proteins, levels of oxidants and markers of oxidative damage and the kinetics of hormone-induced cholesterol transport to mitochondria in adrenal (adrenocortical cells) and Leydig cells isolated from oxidative stress-prone A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX1-/- and Cu,Zn-SOD-/- mice. The second of set studies will focus on measuring changes in these various parameters using adrenal cells and Leydig cells isolated from oxidative stress resistant Cu,Zn-SODTg, Mn-SODTg, and GPX1Tg mice. The final set of studies will examine the impact of simultaneous over-expression of the two antioxidant enzymes on oxidative stress-induced inhibition of steroidogenesis and impaired cholesterol transport to the inner mitochondrial membrane for side-chain cleavage.
Aim 3 is to employ pharmacological strategies to reverse or prevent aging-induced and oxidative stress-associated loss of steroidogenesis. Studies proposed in this section will evaluate the effect of treatment of SAMR-1, SAMP-8, A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX-1-/-, and Cu,Zn-SOD-/-, and control mice, with various low molecular weight antioxidants (NAC, CTMIO, tempol, MnTBAP, EUK-189, ebselen) or p38 MAPK inhibitors (SC-409, SD-169;p38 MAPK is implicated in oxidative stress-mediated inhibition of steroidogenesis) on: a) steroidogenesis;b) mRNA expression of StAR, PBR/TSPO and StarD proteins;c) hormone-induced cholesterol delivery to and with the mitochondria;d) ASK1-p38 MAPK signaling and e) levels of key oxidative stress markers, using adrenal (adrenocortical cells) and testicular Leydig cells.
Steroid hormones are produced by the adrenal gland and gonads. They influence carbohydrate, lipid and protein metabolism, and salt and water balance, as well as immunity and blood pressure, whereas gonadal steroids are critical for secondary sex characteristics, fertility and muscle and bone growth. It is well established that aging in Veterans, like the general population, leads to a decline in testosterone production in men and alteration in adrenal steroid production in both sexes. The goal of this project is to establish the causal role of excessive oxidative stress in age-related decline in steroid hormone production in animal models. Successful completion of these studies may identify potential molecular targets that can be exploited in the development of new therapies to treat aging-induced testosterone deficiency in men and adrenal steroid- related diseases. These studies should also help in understanding the pathogenesis of diabetes, neurodegenerative diseases, and heart disease, where oxidative stress is believed to play an important role.