MicroRNAs, Aging, Cellular Senescence, and Alzheimer's Disease.

Aging is a normal process of living being. It has been reported that multiple cellular changes, including oxidative damage/mitochondrial dysfunction, telomere shortening, inflammation, may accelerate the aging process, leading to cellular senescence. These cellular changes induce age-related human diseases, including Alzheimer's, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, cardiovascular, cancer, and skin diseases. Changes in somatic and germ-line DNA and epigenetics are reported to play large roles in accelerating the onset of human diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent discoveries in molecular biology have revealed that microRNAs (miRNAs) are potential indicators of aging, cellular senescence, and Alzheimer's disease (AD). The purpose of our chapter is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging, cellular senescence, and AD. This chapter also critically evaluates miRNA-based therapeutic drug targets for aging and age-related diseases, particularly Alzheimer's.

Mitochondria-Targeted Molecules as Potential Drugs to Treat Patients With Alzheimer's Disease.

Alzheimer's disease (AD) is the most common multifactorial mental illness affecting the elderly population in the world. Its prevalence increases as person ages. There is no known drug or agent that can delay or prevent the AD and its progression. Extensive research has revealed that multiple cellular pathways involved, including amyloid beta production, mitochondrial structural and functional changes, hyperphosphorylation of Tau and NFT formation, inflammatory responses, and neuronal loss in AD pathogenesis. Amyloid beta-induced synaptic damage, mitochondrial abnormalities, and phosphorylated Tau are major areas of present research investigations. Synaptic pathology and mitochondrial oxidative damage are early events in disease process. In this chapter, a systematic literature survey has been conducted and presented a summary of antioxidants used in (1) AD mouse models, (2) elderly populations, and (3) randomized clinical trials in AD patients. This chapter highlights the recent progress in developing and testing mitochondria-targeted molecules using AD cell cultures and AD mouse models. This chapter also discusses recent research on AD pathogenesis and therapeutics, focusing on mitochondria-targeted molecules as potential therapeutic targets to delay or prevent AD progression.

Androgen metabolites impact CSF amines and axonal serotonin via MAO-A and -B in male macaques.

A number of studies have shown that mutations or deletions of the monoamine oxidase-A (MAO-A) gene cause elevated CNS serotonin and elevated impulsive aggression in humans and animal models. In addition, low cerebrospinal fluid (CSF) 5-hydroxyindole acetic acid (5HIAA) has been documented in a limited number of violent criminal populations and in macaques that exhibit impulsive aggression. To reconcile these different analyses, we hypothesized that CSF 5HIAA reflected degradation of serotonin by the activity of MAO-A; and that low MAO-A activity would result in lower CSF 5HIAA, but overall higher serotonin in the CNS. To test this hypothesis, male Japanese macaques (Macaca fuscata) were castrated, rested for 5-7months, and then treated for 3months with [1] placebo, [2] testosterone (T), [3] dihydrotestosterone (DHT; non-aromatizable androgen) and 1,4,6-androstatriene-3,17-dione (ATD) (steroidal aromatase inhibitor), or [4] flutamide (FLUT; androgen antagonist) and ATD (n=5/group). These treatments enable isolation of androgen and estrogen activities. In the dorsal raphe, MAO-A and MAO-B expressions were determined with in situ hybridization (ISH) and protein expression of aromatase was determined with immunohistochemistry (IHC). CSF concentrations of 5HIAA, 3-methoxy-4-hydroxyphenylglycol (MHPG), and homovanillic acid (HVA) were determined with liquid chromatography/mass spectrometry (LC/MS). From the same animals, previously published data on serotonin axon density were used as a proxy for CNS serotonin. Aromatase conversion of T to estrogen (E) suppressed MAO-A (positive pixel area, p=0.0045), but androgens increased MAO-B (positive pixel area, p=0.014). CSF 5HIAA was suppressed by conversion of T to E (Cohen's d=0.6). CSF 5HIAA was positively correlated with MAO-A-positive pixel area (r(2)=0.78). CSF 5HIAA was inversely correlated with serotonin axon-positive pixel area (r(2)=0.69). In summary, CSF 5HIAA reflects MAO-A activity rather than global serotonin. Low CSF 5HIAA may, in this paradigm, reflect higher serotonin activity. Androgens lower MAO-A activity via metabolism to E, thus elevating CNS serotonin and decreasing CSF 5HIAA. Since androgens increase certain types of aggression, these data are consistent with studies demonstrating that lower MAO-A activity is associated with elevated serotonin and increased aggression.

Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

Depression often accompanies the perimenopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDDs, such as Alzheimer's, Parkinson's, Huntington, amyotrophic lateral sclerosis). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplemented with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus, and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P (estradiol supplemented with progesterone) on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin-proteosome, axon transport and NDD-specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3 per group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and quantitative real-time PCR (qRT-PCR). Increases were confirmed with qRT-PCR in five genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA (proliferating nuclear antigen), GADD45A (DNA damage-inducible), RAD23A (DNA damage recognition) and GTF2H5 (gene transcription factor 2H5) significantly increased with E or E+P treatment (all analysis of variance (ANOVA), P<0.01). Chaperone genes HSP70 (heat-shock protein 70), HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA, P<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA, P<0.003). Transport-related genes coding kinesin, dynein and dynactin increased with E or E+P treatment (all ANOVA, P<0.03). SCNA (α-synuclein) and ADAM10 (α-secretase) increased (both ANOVA, P<0.02) but PSEN1 (presenilin1) decreased (ANOVA, P<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls post hoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA fragmentation. In addition, E or E+P regulated four genes encoding proteins that are often misfolded or malfunctioning in neuronal populations subserving overt NDD symptomology. The expression and regulation of these genes in serotonergic neurons invites speculation that they may mediate an underlying disease process in NDDs, which in turn may be ameliorated or delayed with timely hormone therapy in women.

Effect of inoculum source on the enrichment of microbial communities on two lignocellulosic bioenergy crops under thermophilic and high-solids conditions.

AIMS: Culturing compost-derived microbial communities on biofuel feedstocks under industrial conditions is a technique to enrich for organisms and lignocellulolytic enzymes for bioenergy feedstock deconstruction. In this study, microbial communities from green waste compost (GWC) and grape pomace compost (GPC) were cultured on switchgrass and eucalyptus to observe the impact of inoculation on feedstock decomposition and microbial community structure. METHODS AND RESULTS: Respiration was monitored as a measure of microbial activity, and 16S ribosomal RNA gene sequencing was used to characterize microbial community structure. The enriched community structure and respiration were influenced by the choice of feedstock, compost type, and application of thermophilic, high-solids conditions. However, the effect of compost source was significantly less than the effects of the other culture variables. CONCLUSIONS: Although there are subtle differences in potentially lignocellulolytic taxa between GPC- and GWC-derived communities, these differences do not affect the decomposition rates for these communities on switchgrass or eucalyptus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are useful for designing future experiments to discover lignocellulolytic micro-organisms from compost. They suggest that such work may be better served by deemphasizing screening of compost sources and instead focusing on how compost-derived communities adapt to the feedstocks and process conditions relevant to biofuel production.

Relationships between androgens, serotonin gene expression and innervation in male macaques.

Androgen administration to castrated individuals was purported to decrease activity in the serotonin system. However, we found that androgen administration to castrated male macaques increased fenfluramine-induced serotonin release as reflected by increased prolactin secretion. In this study, we sought to define the effects of androgens and aromatase inhibition on serotonin-related gene expression in the dorsal raphe, as well as serotonergic innervation of the LC. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with (1) placebo, (2) testosterone (T), (3) dihydrotestosterone (DHT; non-aromatizable androgen) and ATD (steroidal aromatase inhibitor), or (4) Flutamide (FLUT; androgen antagonist) and ATD (n=5/group). This study reports the expression of serotonin-related genes: tryptophan hydroxylase 2 (TPH2), serotonin reuptake transporter (SERT) and the serotonin 1A autoreceptor (5HT1A) using digoxigenin-ISH and image analysis. To examine the production of serotonin and the serotonergic innervation of a target area underlying arousal and vigilance, we measured the serotonin axon density entering the LC with ICC and image analysis. TPH2 and SERT expression were significantly elevated in T- and DHT + ATD-treated groups over placebo- and FLUT + ATD-treated groups in the dorsal raphe (p < 0.007). There was no difference in 5HT1A expression between the groups. There was a significant decrease in the pixel area of serotonin axons and in the number of varicosities in the LC across the treatment groups with T > placebo > DHT + ATD = FLUT + ATD treatments. Comparatively, T- and DHT + ATD-treated groups had elevated TPH2 and SERT gene expression, but the DHT + ATD group had markedly suppressed serotonin axon density relative to the T-treated group. Further comparison with previously published data indicated that TPH2 and SERT expression reflected yawning and basal prolactin secretion. The serotonin axon density in the LC agreed with the area under the fenfluramine-stimulated prolactin curve, providing a morphological basis for the pharmacological results. This suggested that androgen activity increased TPH2 and SERT gene expression but, aromatase activity, and neural production of estradiol (E), may subserve axonal serotonin and determination of the compartment acted upon by fenfluramine. In summary, androgens stimulated serotonin-related gene expression, but aromatase inhibition dissociated gene expression from the serotonin innervation of the LC terminal field and fenfluramine-stimulated prolactin secretion.

Hypothalamic KISS1 expression, gonadotrophin-releasing hormone and neurotransmitter innervation vary with stress and sensitivity in macaques.

The present study examined the effect of short-term psychosocial and metabolic stress in a monkey model of stress-induced amenorrhaea on the hypothalamic-pituitary-gonadal axis. KISS1 expression was determined by in situ hybridisation in the infundibular arcuate nucleus. Downstream of KISS1, gonadotrophin-releasing hormone (GnRH) axons in lateral areas rostral to the infundibular recess, serum luteinising hormone (LH) and serum oestradiol were measured by immunohistochemistry and radioimmunoassay. Upstream of KISS1, norepinephrine axons in the rostral arcuate nucleus and serotonin axons in the anterior hypothalamus and periaqueductal grey were measured by immunohistochemistry. Female cynomolgus macaques (Macaca fascicularis) characterised as highly stress resilient (HSR) or stress sensitive (SS) were examined. After characterisation of stress sensitivity, monkeys were either not stressed, or mildly stressed for 5 days before euthanasia in the early follicular phase. Stress consisted of 5 days of 20% food reduction in a novel room with unfamiliar conspecifics. There was a significant increase in KISS1 expression in HSR and SS animals in the presence versus absence of stress (P = 0.005). GnRH axon density increased with stress in HSR and SS animals (P = 0.015), whereas LH showed a gradual but nonsignificant increase with stress. Oestradiol trended higher in HSR animals and there was no effect of stress (P = 0.83). Norepinephrine axon density (marked with dopamine ß-hydroxylase) increased with stress in both HSR and SS groups (P ≤ 0.002), whereas serotonin axon density was higher in HSR compared to SS animals and there was no effect of stress (P = 0.03). The ratio of dopamine ß-hydroxylase/oestradiol correlated with KISS1 (P = 0.052) and GnRH correlated with serum LH (P = 0.039). In conclusion, oestradiol inhibited KISS1 in the absence of stress, although stress increased norepinephrine, which may over-ride oestradiol inhibition of KISS1 expression. We speculate that neural pathways transduce stress to KISS1 neurones, which changes their sensitivity to oestradiol.

Bilateral arterial variation in the upper extremity--an anatomical case report.

We report a case of bilateral arterial variation in the upper extremities of a male cadaver. In the left upper extremity, it was observed that the ulnar artery was arising from axillary artery. This ulnar artery was superficial throughout its course at the medial aspect of the arm and forearm. It was highly tortuous and did not have any branches either in the arm or forearm. It ended as the superficial palmar arch in the palm. However the brachial artery entered the cubital fossa and divided into radial and common interosseus arteries. In contrast, the right upper extremity was having normal brachial artery which was terminated into the ulnar and radial arteries at the cubital fossa. However, there was an accessory brachial artery present at the arm and ended at the cubital fossa. We believe that these anatomical variations are because of abnormal developmental vascular pattern in the region. In clinical practice, accurate knowledge of the arterial variations of upper extremity is of considerable importance in case of reparative surgeries and fracture management. They are of interest to the vascular and plastic surgeons.

Thermophilic enrichment of microbial communities in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate.

AIMS: The aim of the study was to develop an approach to enrich ionic liquid tolerant micro-organisms that efficiently decompose lignocellulose in a thermophilic and high-solids environment. METHODS AND RESULTS: High-solids incubations were conducted, using compost as an inoculum source, to enrich for thermophilic communities that decompose switchgrass in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]). Ionic liquid levels were increased from 0 to 6% on a total weight basis incrementally. Successful enrichment of a community that decomposed lignocellulose at 55°C in the presence of 6% [C2mim][OAc] was achieved, when the [C2mim][OAc] level was increased stepwise from 2% to 4% to 5% to 6%. Pyrosequencing results revealed a shift in the community and a sharp decrease in richness, when thermophilic conditions were applied. CONCLUSIONS: A community tolerant to a thermophilic, high-solids environment containing 6% [C2mim][OAc] was enriched from compost. Gradually increasing [C2mim][OAc] concentrations allowed the community to adapt to [C2mim][OAc]. SIGNIFICANCE AND IMPACT OF THE STUDY: A successful approach to enrich communities that decompose lignocellulose under thermophilic high-solids conditions in the presence of elevated levels of [C2mim][OAc] has been developed. Communities yielded from this approach will provide resources for the discovery of enzymes and metabolic pathways relevant to biomass pretreatment and fuel production.

Swyer syndrome: a case report with literature review.

Gonadoblastomas are known to develop in dysgenetic gonads, especially so, if Y chromosome material is present. A 20-years-old girl who noticed breast development since the age of 12 years presented with primary amenorhoea, distension of lower abdomen and intermittent pain for two months. She had breakthrough bleeding with six months of estrogen replacement. Tanner breast stage was five and pubic hair stage was also five. Examination revealed a mass in the lower abdomen extending into hypogastrium, umbilical and lumbar regions. Her gonadotropin levels were grossly elevated. Karyotyping showed 46XY. CT scan of abdomen showed a 17X11 cm mass in the pelvis without visible gonads. Surgical excision of the mass along with bilateral salpingophorectomy was performed. Histopathology revealed the mass to be a dysgerminoma, while the right gonad lodged gonadoblastoma. She was diagnosed as a rare case of Swyer syndrome.

Long-term ovariectomy decreases serotonin neuron number and gene expression in free ranging macaques.

The serotonin system responds to the ovarian steroids, estradiol (E) and progesterone (P), in women and female animal models. In macaques, ovarian steroid administration to ovariectomized (Ovx) individuals improves serotonin neural function through actions on pivotal serotonin-related genes and proteins, such as TPH2 (tryptophan hydroxylase 2), SERT (serotonin reuptake transporter), and the 5HT1A autoreceptor. In addition, ovarian steroid administration reduces gene and protein expression in the caspase-independent pathway and reduces DNA fragmentation in serotonin neurons. This study examines the hypothesis that long-term ovariectomy will lead to a loss of serotonin neurons and compromised gene expression in serotonin neurons. Female Japanese macaques were ovariectomized or tubal ligated (n=5/group) at 3 years of age and returned to their natal troop. After 3 years, the animals were collected, administered a fenfluramine challenge to determine global serotonin availability, and then euthanized. Fev, TPH2, SERT, and 5HT1A expression were examined with digoxigenin in situ hybridization (ISH) and quantitative image analysis. Cell number, positive pixel area, and average pixel density were determined. In the Ovx group, Fev, TPH2, SERT, and 5HT1A showed a significant decease in average and total cell number and positive pixel area. The reduction in Fev-positive neurons suggests that there were fewer serotonin neurons in Ovx animals compared to ovary-intact animals. The decrease in TPH2 in the Ovx animals was consistent with earlier results in 5-month Ovx animals, but it may be due to the decrease in cell number rather than a decrease in expression on an individual cell basis. The decrease in SERT and 5HT1A in long-term Ovx differed from previous studies in short-term Ovx. In summary, long-term ovarian steroid loss resulted in fewer serotonin neurons and overall lower Fev, TPH2, SERT, and 5HT1A gene expression. This may be due to serotonin cell death or to a negative impact on a long-term developmental process in young female macaques.

Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate.

AIMS: This work aimed to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. METHODS AND RESULTS: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. CONCLUSIONS: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.

Ovarian steroid regulation of the midbrain corticotropin releasing factor and urocortin systems in macaques.

A significant number of postmenopausal women report increased anxiety and vulnerability to stress, which has been linked to decreased secretion of ovarian steroids. Communication between the serotonin system and the corticotropin releasing factor (CRF) system determines stress sensitivity or resilience. This study examines the effects of the ovarian steroids, estradiol (E) and progesterone (P) on the CRF system components that impact serotonin neurons in the midbrain of nonhuman primates. Ovariectomized rhesus macaques were treated with placebo, E alone for 1 month, or E supplemented with P for the last 2 weeks. Quantitative (q)RT-PCR and immunocytochemistry were employed. E±P treatment decreased CRF-R1 and increased CRF-R2 gene expression in hemi-midbrain blocks and in laser captured serotonin neurons. Also in hemi-midbrains, E treatment increased urocortin 1 (UCN1) and CRFBP gene expression, but supplemental P treatment reversed these effects. E±P decreased CRF fiber density in the dorsal, interfascicular and median raphe nuclei and decreased CRF-R1 immunostaining in the dorsal raphe. E increased CRF-R2 immunostaining in the dorsal and median raphe. E±P increased UCN1 immunostaining in the cell bodies and increased UCN1 fiber density in the caudal linear nucleus. Estrogen receptor beta (ERß), but not ERα was detected in the nucleus of UCN1-positive neurons. While the mechanism of ovarian hormone regulation of the midbrain CRF system requires further investigation, these studies clearly demonstrate another pathway by which ovarian hormones may have positive effects on anxiety and mood regulation.

Effect of ovarian hormones on genes promoting dendritic spines in laser-captured serotonin neurons from macaques.

Dendritic spines are the elementary structural units of neuronal plasticity and the cascades that promote dendritic spine remodeling center on Rho GTPases and downstream effectors of actin dynamics. In a model of hormone replacement therapy, we sought the effect of estradiol (E) and progesterone (P) on gene expression in these cascades in laser-captured serotonin neurons from rhesus macaques with complementary DNA array analysis. Ovariectomized rhesus macaques were treated with either placebo, E or E+P through Silastic implant for 1 month before euthanasia. The midbrain was obtained, sectioned and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (PixCell II). RNA from laser-captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips. With E±P treatment, there was a significant change in 744 probe sets (analysis of variance, P<0.05), but 10,493 probe sets exhibited a twofold or greater change. Pivotal changes in pathways leading to dendritic spine proliferation and transformation included twofold or greater increases in expression of the Rho GTPases called CDC42, Rac1 and RhoA. In addition, twofold or greater increases occurred in downstream effectors of actin dynamics, including p21-activated kinase (PAK1), Rho-associated coiled-coil-containing protein kinase (ROCK), PIP5K, IRSp53, Wiskott-Aldrich syndrome protein (WASP), WASP family Verprolin-homologous protein (WAVE), MLC, cofilin, gelsolin, profilin and three subunits of actin-related protein (ARP2/3). Finally, twofold or greater decreases occurred in CRIPAK, LIMK2 and myosin light chain kinase (MLCK). The regulation of RhoA, Rac1, CDC42, ROCK, PIP5k, IRSp53, WASP, WAVE, LIMK2, CRIPAK1, MLCK, ARP2/3 subunit 3, gelsolin, profilin and cofilin was confirmed with nested quantitative reverse transcriptase-PCR on laser-captured RNA (n=3 animals/treatment). The data indicate that ovarian steroids target gene expression of the Rho GTPases and pivotal downstream proteins, that in turn would promote dendritic spine proliferation and stabilization on serotonin neurons of the dorsal raphe nucleus.

Stress sensitive female macaques have decreased fifth Ewing variant (Fev) and serotonin-related gene expression that is not reversed by citalopram.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60 days or two menstrual cycles, highly stress resilient monkeys (HSR) continued to ovulate during the stress cycles whereas stress sensitive monkeys (SS) did not. After cessation of stress, monkeys characterized as HSR or SS were administered placebo (PL) or S-citalopram (CIT) for 15 weeks at doses that normalized ovarian steroid secretion in the SS animals and that maintained blood CIT levels in a therapeutic range. After euthanasia, the brain was perfused with 4% paraformaldehyde. The pontine midbrain was blocked and sectioned at 25 microm. The expression of four genes pivotal to serotonin neural function was assessed in the four groups of monkeys (n=4/group). Fev (fifth Ewing variant) ETS transcription factor, tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor were determined at 7-8 levels of the dorsal raphe nucleus with in situ hybridization (ISH) using radiolabeled- and digoxygenin-incorporated riboprobes. Positive pixel area and cell number were measured with Slidebook 4.2 in the digoxigenin assay for Fev. Optical density (OD) and positive pixel area were measured with NIH Image software in the radiolabeled assays for TPH2, SERT and 5HT1A. All data were analyzed with two-way ANOVA. SS monkeys had significantly fewer Fev-positive cells and lower Fev-positive pixel area in the dorsal raphe than HSR monkeys. SS monkeys also had significantly lower levels of TPH2, SERT and 5HT1A mRNAs in the dorsal raphe nucleus than HSR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. These data suggest that SS monkeys have fewer serotonin (5-HT) neurons than HSR monkeys, and that they have deficient Fev expression, which in turn, leads to deficient TPH2, SERT and 5HT1A expression. In addition, the therapeutic effect of CIT is probably achieved through mechanisms other than alteration of 5-HT-related gene expression.

Unique responses of midbrain CART neurons in macaques to ovarian steroids.

CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger-Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys (Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+P. Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR (n=3/group); (b) for immunohistochemical analysis of CART and CART+tryptophan hydroxylase (TPH), CART+estrogen receptors (ER) or CART+progesterone receptors (n=5/group) and (c) for Western blots (n=3/group). Both E- and E+P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger-Westphal nucleus indicated little effect of E or E+P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student's t-test, but not with ANOVA. CART 55-102 detection on Western blot was unchanged by hormone administration. ERbeta and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

Neuroprotective actions of ovarian hormones without insult in the raphe region of rhesus macaques.

Using a nonhuman primate model of surgical menopause, our laboratory has shown that ovarian hormone treatment (HT) improves 5-HT neural function in the dorsal raphe nucleus (DRN). We further hypothesize that HT may increase 5-HT neuronal resilience. Recent data from microarray analysis indicated that HT regulates gene expression in pathways that lead to apoptosis. In this study, we questioned whether HT alters protein expression in caspase-dependent and independent pathways. Ovariectomized monkeys received Silastic implants containing placebo (empty), estrogen (E) or E+ progesterone (P). A small block of the midbrain containing the DRN was dissected and subjected to subcellular fractionation, yielding cytosolic, nuclear and mitochondrial fractions (n=4/group). The pro-apoptotic protein, c-jun n-terminal kinase (JNK1) and its phosphorylation were decreased by E+P treatment in the cytosolic fraction. Downstream of JNK are proteins in the caspase-dependent and -independent pathways. First, in the caspase-dependent pathway, cytoplasmic and mitochondrial fractions were immunoblotted for Bcl-2 family members, cytochrome c, Apaf1 and XIAP. However, the expression of these proteins did not differ among treatments. Pro-caspase 3 was decreased by E+P, but there was no evidence of active caspase in any group. Then, we examined the involvement of a protein in the caspase-independent pathway, called apoptosis-inducing factor (AIF). AIF mRNA (n=3/group) and AIF mitochondrial protein tended to decrease with hormone treatment. However, AIF protein in the nuclear fraction in E+P treated monkeys was significantly reduced. This indicates that HT is reducing the translocation of AIF from mitochondria to nucleus, thus inhibiting AIF-mediated apoptosis. AIF was immunocytochemically localized to large 5-HT-like neurons of the dorsal raphe. These data suggest that in the absence of global trauma or ischemia, HT may act through the caspase-independent pathway to promote neuroprotection in the 5-HT system.

Serotonin-related gene expression in female monkeys with individual sensitivity to stress.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. In this study, the expression of four genes pivotal to serotonin neural function was assessed in monkeys previously categorized as highly stress resistant (n=3; normal menstrual cyclicity through two stress cycles), medium stress resistant (n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e. stress-sensitive; n=4; anovulatory as soon as stress is initiated). In situ hybridization and quantitative image analysis was used to measure mRNAs coding for SERT (serotonin transporter), 5HT1A autoreceptor, MAO-A and MAO-B (monoamine oxidases) at six levels of the dorsal raphe nucleus (DRN). Optical density (OD) and positive pixel area were measured with NIH Image software. In addition, serotonin neurons were immunostained and counted at three levels of the DRN. Finally, each animal was genotyped for the serotonin transporter long polymorphic region (5HTTLPR). Stress sensitive animals had lower expression of SERT mRNA in the caudal region of the DRN (P<0.04). SERT mRNA OD in the caudal DRN was positively correlated with serum progesterone during a pre-stress control cycle (P<0.0007). 5HT1A mRNA OD signal tended to decline in the stress-sensitive group, but statistical difference between averages was lacking in analysis of variance. However, 5HT1A mRNA signal was positively correlated with control cycle progesterone (P<0.009). There was significantly less MAO-A mRNA signal in the stress-sensitive group (P<0.007) and MAO-A OD was positively correlated with progesterone from a pre-stress control cycle (P<0.007). MAO-B mRNA exhibited a similar downward trend in the stress-sensitive group. MAO-B OD also correlated with control cycle progesterone (P<0.003). There were significantly fewer serotonin neurons in the stress-sensitive group. All animals contained only the long form of the 5HTTLPR. Thus, all serotonin-related mRNAs examined in the dorsal raphe to date were lower (SERT, MAO-A) or exhibited a lower trend (5HT1A, MAO-B) in the stress sensitive animals, which probably reflects the lower number of serotonin neurons present.

Application of matrix solid-phase dispersion in the determination of dibenzo[a,l]pyrene content of experimental animal diets used in a large-scale tumor study.

A method utilizing matrix solid-phase dispersion (MSPD) was developed for isolation and determination of dibenzo[a,l]pyrene (DBP) in experimental rainbow-trout diets used in a large-scale carcinogenesis study. A 0.5 g sample of moist ration containing 0-225 ppm DBP (dry basis) was mixed with 2 g C18 sorbent and benzo[a]pyrene internal standard was added to the mixture. Extraction and clean-up were accomplished in a single step by extracting the sample mixture with hexane-benzene 4:1 from a cartridge containing 2 g Florisil. DBP was quantified by HPLC on a C5 bonded phase column with fluorescence detection. Mean analytical recovery of DBP from control diet spiked at three concentration levels was 101 to 107% with relative standard deviations of 1 to 7%. The limit of detection of DBP was equivalent to 0.014 ppm in the ration. Application of the method to verification of DBP levels in trout rations from the carcinogenesis study is described. Control ration (0 ppm DBP) was screened for possible DBP contamination and none was found. This is the first report on analysis of DBP in experimental animal diets.

Marker assisted selection of bacterial blight resistance genes in rice.

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.