Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure.

A typical clinical cis-diamminedichloroplatinum(II) (cisplatin) dosing regimen consists of repeated treatment cycles followed by a recovery period. While effective, this dosing structure results in a prolonged, often permanent, infertility in men. Spermatogonial stem cells (SSCs) are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. We propose that an altered spermatogonial environment during recovery from the initial treatment cycle drives an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced injury from subsequent cycles. To test this hypothesis, the undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery periods of a clinically-relevant cisplatin exposure paradigm. Histological examination revealed a disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) staining indicated an increase in apoptotic frequency following exposure. Immunohistochemical examination of Foxo1 and incorporated BrdU showed an increase in the undifferentiated spermatogonial population and mitotic activity in the recovery period in mice exposed to one cycle, but not two cycles of cisplatin. Immunohistochemical investigation of glial cell line-derived neurotrophic factor (GDNF) revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.

Multisensory convergence of visual and haptic object preference across development.

Visuohaptic inputs offer redundant and complementary information regarding an object׳s geometrical structure. The integration of these inputs facilitates object recognition in adults. While the ability to recognize objects in the environment both visually and haptically develops early on, the development of the neural mechanisms for integrating visual and haptic object shape information remains unknown. In the present study, we used functional Magnetic Resonance Imaging (fMRI) in three groups of participants, 4 to 5.5 year olds, 7 to 8.5 year olds, and adults. Participants were tested in a block design involving visual exploration of two-dimensional images of common objects and real textures, and haptic exploration of their three-dimensional counterparts. As in previous studies, object preference was defined as a greater BOLD response for objects than textures. The analyses specifically target two sites of known visuohaptic convergence in adults: the lateral occipital tactile-visual region (LOtv) and intraparietal sulcus (IPS). Results indicated that the LOtv is involved in visuohaptic object recognition early on. More importantly, object preference in the LOtv became increasingly visually dominant with development. Despite previous reports that the lateral occipital complex (LOC) is adult-like by 8 years, these findings indicate that at least part of the LOC is not. Whole-brain maps showed overlap between adults and both groups of children in the LOC. However, the overlap did not build incrementally from the younger to the older group, suggesting that visuohaptic object preference does not develop in an additive manner. Taken together, the results show that the development of neural substrates for visuohaptic recognition is protracted compared to substrates that are primarily visual or haptic.

Chronic 17beta-estradiol or cholesterol prevents stress-induced hippocampal CA3 dendritic retraction in ovariectomized female rats: possible correspondence between CA1 spine properties and spatial acquisition.

Chronic stress may have different effects on hippocampal CA3 and CA1 neuronal morphology and function depending upon hormonal status, but rarely are manipulations of stress and gonadal steroids combined. Experiment 1 investigated the effects of chronic restraint and 17beta-estradiol replacement on CA3 and CA1 dendritic morphology and spatial learning in ovariectomized (OVX) female Sprague-Dawley rats. OVX rats were implanted with 25% 17beta-estradiol, 100% cholesterol, or blank silastic capsules and then chronically restrained (6h/d/21d) or kept in home cages. 17beta-Estradiol or cholesterol prevented stress-induced CA3 dendritic retraction, increased CA1 apical spine density, and altered CA1 spine shape. The combination of chronic stress and 17beta-estradiol facilitated water maze acquisition compared to chronic stress + blank implants and nonstressed controls + 17beta-estradiol. To further investigate the interaction between 17beta-estradiol and stress on hippocampal morphology, experiment 2 was conducted on gonadally intact, cycling female rats that were chronically restrained (6h/d/21d), and then euthanized at proestrus (high ovarian hormones) or estrus (low ovarian hormones). Cycling female rats failed to show chronic stress-induced CA3 dendritic retraction at either estrous phase. Chronic stress enhanced the ratio of CA1 basal spine heads to headless spines as found in experiment 1. In addition, proestrous rats displayed increased CA1 spine density regardless of stress history. These results show that 17beta-estradiol or cholesterol protect against chronic stress-induced CA3 dendritic retraction in females. These stress- and 17beta-estradiol-induced morphological changes may provide insight into how dendritic complexity and spine properties contribute to spatial ability.

Chronic glucocorticoids increase hippocampal vulnerability to neurotoxicity under conditions that produce CA3 dendritic retraction but fail to impair spatial recognition memory.

We previously found that chronic stress conditions producing CA3 dendritic retraction and spatial memory deficits make the hippocampus vulnerable to the neurotoxin ibotenic acid (IBO). The purpose of this study was to determine whether exposure to chronic corticosterone (CORT) under conditions that produce CA3 dendritic retraction would enhance CA3 susceptibility to IBO. Male Sprague Dawley rats were chronically treated for 21 d with CORT in drinking water (400 microg/ml), and half were given daily injections of phenytoin (40 mg/kg), an antiepileptic drug that prevents CA3 dendritic retraction. Three days after treatments stopped, IBO was infused into the CA3 region. Conditions producing CA3 dendritic retraction (CORT and vehicle) exacerbated IBO-induced CA3 damage compared with conditions in which CA3 dendritic retraction was not observed (vehicle and vehicle, vehicle and phenytoin, CORT and phenytoin). Additionally, spatial recognition memory was assessed using the Y-maze, revealing that conditions producing CA3 dendritic retraction failed to impair spatial recognition memory. Furthermore, CORT levels in response to a potentially mild stressor (injection and Y-maze exposure) stayed at basal levels and failed to differ among key groups (vehicle and vehicle, CORT and vehicle, CORT and phenytoin), supporting the interpretations that CORT levels were unlikely to have been elevated during IBO infusion and that the neuroprotective actions of phenytoin were not through CORT alterations. These data are the first to show that conditions with prolonged glucocorticoid elevations leading to structural changes in hippocampal dendritic arbors can make the hippocampus vulnerable to neurotoxic challenges. These findings have significance for many disorders with elevated glucocorticoids that include depression, schizophrenia, Alzheimer's disease, and Cushing's disease.

CRP subunit association and hinge conformation changes in response to cAMP binding: analysis of C-helix cysteine-substituted CRP.

We investigated the characteristics of 13 CRP variants having cysteine substituted at positions 113, 115, 116, 117, 118, 120, 122, 124, 126, 127, 129, 130, or 131, positions that span the length of the CRP C alpha-helix. Under reducing conditions, the WT and all Cys-substituted forms of CRP migrated as 23.5 kDa CRP monomer species on SDS-PAGE gels. In the absence of a reductant, 9 of 13 Cys-substituted forms of CRP including the L113C, S117C, M120C, L124C, V126C, T127C, E129C, K130C, and V131C CRP contained protein that migrated as 47 kDa CRP dimer species on SDS-PAGE gels. CNBr digestion of the protein preparations followed by MALDI-TOF MS analysis of the peptide fragments showed these 47 kDa species to be CRP dimers that originated from disulfide bonds formed between positional-pair C alpha-helix Cys residues. The ratio of monomer CRP and disulfide cross-linked CRP within a Cys-substituted CRP preparation was found to be independent of cAMP for Cys-substituted CRP preparations denatured and renatured in the presence of various cAMP concentrations. This finding suggests that there is no large-scale concerted motion (i.e., scissoring) of the CRP subunits in response to cAMP binding. In addition, we have identified three amino acid residues located along the CRP C alpha-helix that play a role in facilitating the conformation transition of the CRP hinge from that characteristic of apo-CRP to that characteristic of the CRP.cAMP complex.

Attenuating corticosterone levels on the day of memory assessment prevents chronic stress-induced impairments in spatial memory.

This study investigated whether chronic stress-induced spatial memory deficits were caused by changes in the hypothalamic-pituitary-adrenal axis, such as corticosterone (CORT) elevations on the day of memory assessment, rather than the consequence of structural changes in the hippocampus. Male Sprague-Dawley rats were restrained for 6 h/day/21 days, and spatial memory was assessed on the Y-maze on day 22. Ninety minutes before training, rats received a subcutaneous injection of vehicle or metyrapone, a CORT synthesis inhibitor, and then spatial memory was determined 4-h later. The highest dose of metyrapone (75 mg/kg, s.c.) was most effective at preventing stress-induced spatial memory deficits. Chronic stress increased total CORT levels following Y-maze exposure, while acute metyrapone treatment dose-dependently attenuated total and free (unbound) CORT levels in both stress and control conditions. Blood samples taken from a separate subset of chronically stressed rats showed that baseline CORT levels were similar across the restraint period. Finally, chronic stress down-regulated glucocorticoid, but not mineralocorticoid, receptor mRNA expression within the hippocampus (dentate gyrus, CA1, CA2, CA3). These findings suggest that chronic stress-induced spatial memory deficits may be mediated by hypothalamic-pituitary-adrenal axis dysregulation. Specifically, CORT elevations and reductions in hippocampal glucocorticoid receptor expression, at the time of behavioural assessment may be involved, as opposed to a direct effect that is solely dependent upon hippocampal structural changes. These results have significance for treating cognitive decline in conditions associated with elevated glucocorticoids that include subpopulations in ageing, depression, Cushing's disease and Alzheimer's disease.

Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli.

Expression of catabolite-sensitive operons in Escherichia coli is cAMP-dependent and mediated through the CRP:cAMP complex binding to specific sequences in DNA. Five specific ionic or polar interactions occur in cAMP binding pocket of CRP. E72 interacts with the cAMP 2' OH, R82 and S83 interact with the negatively charged phosphate moiety, and T127 and S128 interact with the adenine ring. There is evidence to suggest that E72 and R82 may mediate an essential CRP molecular switch mechanism. Therefore, stimulation of CRP transcription activation was examined by perturbing these residues. Further, CRP:cAMP complex was treated with a specific DNA sequence containing the lac CRP binding site along with RNA polymerase to mimic in vivo conditions. Biochemical and biophysical results revealed that regulation of transcription activation depends on alignment of CRP tertiary structure through inter-domain communication and it was concluded that positions 72 and 82 are essential in the activation of CRP by cAMP.

Acute stress impairs spatial memory in male but not female rats: influence of estrous cycle.

We investigated how sex and estrous cycle influenced spatial recognition memory in the Y-maze after exposure to acute restraint stress. In Experiment 1, intact male and female rats were restrained for 1 h and then 2 h after the start of restraint, rats were trained on the Y-maze. After a 4 h delay, hippocampal-dependent spatial recognition memory was assessed. Acute stress produced opposite patterns between the sexes with spatial memory being impaired in males and facilitated in females. Serum corticosterone measures indicated that both sexes showed a robust corticosterone response after restraint and a moderate corticosterone response after Y-maze exposure. Serum corticosterone levels in response to restraint and Y-maze were not statistically different between the sexes. Experiment 2 examined the influence of the estrous cycle on spatial memory ability after acute stress. Acute stress facilitated spatial memory in females compared to controls, regardless of the estrous cycle phase (estrus and proestrus). Moreover, females in proestrus showed higher serum corticosterone levels during restraint compared to females in estrus. No differences in corticosterone levels were observed at baseline or following 2 h of recovery from restraint. These data show important differences in how sex and estrous cycle influence cognitive functions following acute stress.

Interaction of CRP L124 with cAMP affects CRP cAMP binding constants, cAMP binding cooperativity, and CRP allostery.

A cyclic nucleotide-binding pocket of the CRP dimer is composed of amino acid residues contributed by both subunits. Leucine (L) 124 of one subunit packs against the adenine ring of cAMP bound to the opposing subunit. We have undertaken a study designed to evaluate the role of L124 in CRP allostery. Wild-type (WT) apo-CRP is a 47 kDa protease-resistant dimer composed of identical subunits that exhibits a biphasic isotherm in cAMP titration studies. The WT CRP-cAMP complex is a protease-sensitive dimer degraded by protease to a dimer core that ranges between 26.5 and 30.5 kDa. Substitution of L124 with isoleucine (I), valine (V), cysteine (C), or alanine (A) generated a series of CRP variants that exhibited unique differences in apo-CRP resistance to protease, the mass of the core fragments generated in protease digestion reactions, cAMP-mediated allostery, and CRP-cAMP complex functionality. Differences in the affinity of the position 124 CRP variants for cAMP were observed. The binding constants that drive the formation of the WT and L124I CRP-cAMP complexes deviated by not more than a factor of 1.5. In contrast, the L124V, L124A, and L124C forms of CRP exhibited both a decreased K(cAMP1)(app) and an increased K(cAMP2)(app) to produce 2.4-, 55-, and 204-fold reductions, respectively, in the difference between these two parameters compared to that observed for WT CRP. The data indicate that the van der Waals volume and/or the hyrophobicity of the L124 side chain are important determinants of CRP cAMP binding properties and affect, either directly or indirectly, cAMP-mediated conformation changes in CRP.