Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable In Vivo Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.

Monitoring of chronic wasting disease (CWD) (IV).

The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017-28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease-associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high-risk target groups for surveillance should be revised, and 'road kill' removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high-risk target groups, sustained over time, using sampling units and a data-driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data-driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double-strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.

Apparent stability masks underlying change in a mule deer herd with unmanaged chronic wasting disease.

The contagious prion disease "chronic wasting disease" (CWD) infects mule deer (Odocoileus hemionus) and related species. Unchecked epidemics raise ecological, socioeconomic, and public health concerns. Prion infection shortens a deer's lifespan, and when prevalence (proportion of adults infected) becomes sufficiently high CWD can affect herd dynamics. Understanding population responses over time is key to forecasting long-term impacts. Here we describe unexpected stability in prevalence and abundance in a mule deer herd where CWD has been left unmanaged. High apparent prevalence (~30%) since at least 2005 likely drove observed changes in the proportion and age distribution of wild-type native prion protein (PRNP) gene homozygotes among deer sampled. Predation by mountain lions (Puma concolor) may be helping keep CWD in check. Despite stable appearances, prion disease nonetheless impairs adult survival and likely resilience in this deer herd, limiting its potential for growth despite refuge from hunter harvest and favorable habitat and winter conditions.

MOUNTAIN LIONS (PUMA CONCOLOR) RESIST LONG-TERM DIETARY EXPOSURE TO CHRONIC WASTING DISEASE.

For nearly 18 yr, we evaluated susceptibility of captive mountain lions (Puma concolor) to chronic wasting disease (CWD) in the face of repeated exposure associated with consuming infected cervid carcasses. Three mountain lions with a monomorphic prion protein gene (PRNP) sequence identical to that described previously for the species had access to parts of ≥432 infected carcasses during ≥2,013 feeding occasions, conservatively representing >14,000 kg of infected feed material, during May 2002 to March 2020. The proportion of diet in infected carcass material averaged 43% overall but differed from year to year (minimally 11-74%). Most infected carcasses were mule deer (Odocoileus hemionus; ∼75%). We observed no clinical signs suggestive of progressive encephalopathy or other neurologic disease over the ∼14.5-17.9 yr between first known exposure and eventual death. Histopathology revealed no spongiform changes or immunostaining suggestive of prion infection in multiple sections of nervous and lymphoid tissue. Similarly, none of 133 free-ranging mountain lion carcasses sampled opportunistically during 2004-20 showed immunostaining consistent with prion infection in sections of brainstem or lymph node. These findings align with prior work suggesting that CWD-associated prions face strong barriers to natural transmission among species outside the family Cervidae.

Reduction of Chronic Wasting Disease Prion Seeding Activity following Digestion by Mountain Lions.

Chronic wasting disease (CWD) is a transmissible prion disease first observed in the 1960s in North America. This invariably fatal disease affects multiple cervid species in the wild and in captivity. In addition to the several known transmission pathways involving cervid host species, prions have been detected in the feces of crows and coyotes after consumption of experimentally spiked tissues. This raises questions about the role of cervid consumers in the perpetuation of CWD. Mountain lions have been shown to preferentially select CWD-infected prey and are also apparently resistant to infection. In this study, two captive mountain lions were fed ground mule deer muscle tissue spiked with brain-derived CWD prions, and lion feces were collected for 1 week afterward. The input brain and resulting fecal materials were analyzed using the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to quantify prion seeding activity. We recovered only 2.8 to 3.9% of input CWD prions after passage through the mountain lions' gastrointestinal tracts. Interestingly, CWD prions were shed only in the first defecation following consumption. Our data support the possibility that mountain lions feeding upon infected carcasses could excrete CWD prions in their feces over a short period of time but also suggest that most of the ingested prions are eliminated or sequestered by this large predator. IMPORTANCE CWD prions appear to spread naturally among susceptible cervid species in captivity and in the wild. A better understanding of all the ways these prions move, persist, and subsequently infect target species through the environment is critical to developing comprehensive disease control strategies. In our study, we show limited, transient pass-through of CWD prions in an apex predator, the mountain lion, using the highly sensitive RT-QuIC assay on feces collected after lions were fed prion-spiked muscle tissue. Prions were detected in feces only in the first defecation after exposure. Moreover, the amount of CWD prions recovered in feces was reduced by >96% after passing through the lion digestive system. This indicates that mountain lions may have some potential to distribute CWD prions within their home ranges but that they also effectively eliminate most of the CWD prions they consume.

THE RELATIONSHIP BETWEEN HARVEST MANAGEMENT AND CHRONIC WASTING DISEASE PREVALENCE TRENDS IN WESTERN MULE DEER (ODOCOILEUS HEMIONUS) HERDS.

We analyzed retrospective data on harvest management practices and corresponding chronic wasting disease (CWD) prevalence trends in 36 western US and Canadian mule deer (Odocoileus hemionus) management units (units). Our analyses employed logistic regression and model selection, exploiting variation in practices within and among jurisdictions to examine relationships between harvest management and apparent prevalence (the proportion of positive animals among those sampled). Despite notable differences in hunting practices among jurisdictions, our meta-analysis of combined data revealed strong evidence that the amount of harvest was related to CWD prevalence trends among adult male mule deer in the 32 units where prevalence at the start of the analysis period was ≤5%. All competitive models included the number of male deer harvested or number of hunters 1-2 yr prior as an explanatory variable, with increasing harvest leading to lower prevalence among males harvested in the following year. Competitive models also included harvest timing. Although less definitive than the number harvested, median harvest dates falling closer to breeding seasons were associated with lower prevalence in the following year. Our findings suggest harvest-when sufficient and sustained-can be an effective tool for attenuating CWD prevalence in adult male mule deer across western ranges, especially early in the course of an epidemic. Evidence of a broad relationship between the amount of harvest and subsequent changes in CWD prevalence among adult male mule deer provides an empirical basis for undertaking adaptive disease management experimentation aimed at suppressing or curtailing CWD epidemics.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Inferring Chronic Wasting Disease Incidence from Prevalence Data.

Incidence of chronic wasting disease infection showed strong, positive correlation (r≥0.944) with apparent prevalence among female and male mule deer (Odocoileus hemionus) in seven herds previously studied in Colorado and Wyoming, US. With attention to monitoring method consistency and context, inferring that observed prevalence trends reflect underlying epidemic dynamics in mule deer herds appears justifiable.

EVALUATING THE USE OF A BUTORPHANOL-AZAPERONE-MEDETOMIDINE FIXED-DOSE COMBINATION FOR STANDING SEDATION IN AFRICAN ELEPHANTS (LOXODONTA AFRICANA).

This study investigated the use of a fixed-dose combination of 30 mg/ml butorphanol, 12 mg/ml azaperone, and 12 mg/ml medetomidine for the standing sedation of captive African elephants (Loxodonta africana). In total, seven females (mean age 19.6 yr; range 6-31 yr) and six males (mean age 33.5 yr; range 9-35 yr) were sedated. The estimated dose was 0.0005 ± 0.0001 ml/kg and 0.006 ± 0.001 ml/cm shoulder height, which resulted in a dose of 0.016 ± 0.002 mg/kg or 0.19 ± 0.04 mg/cm shoulder height butorphanol, 0.006 ± 0.0008 mg/ kg or 0.076 ± 0.015 mg/cm shoulder height azaperone, and 0.006 ± 0.0008 mg/kg or 0.076 ± 0.015 mg/cm medetomidine. First signs of sedation were observed within 3-10 min (mean 6 ± 2 min) after darting, and monitoring of the animals started on average at 24 ± 9 min after darting. No bradycardia was observed in any of the elephants (mean heart rate 40.0 ± 6.55 beats/min), although all the animals were mildly hypotensive (mean blood pressure 118.5/86 [94.5]). Rectal temperatures fell within acceptable ranges, and respiratory parameters were stable in all the animals throughout sedation and fell within the standard ranges reported for conscious, standing elephants. Only one elephant had clinically significant hypoxemia characterized by a partial pressure of oxygen (PaO2) < 60 mm Hg. This elephant was also hypercapnic (PaCO2 > 50 mm Hg), although pH and peripheral capillary oxygen saturation fell within acceptable ranges. None of the elephants reacted to moderately painful stimuli while sedated. The combination was reversed with intramuscular injections of naltrexone (1 mg for every 1 mg butorphanol) and atipamezole (5 mg for every 1 mg medetomidine). Recovery was smooth and calm in all the animals. Time from injection of the reversals until the first signs of recovery was 4.6 ± 2.01 min (range 1-8 min).

Detection of chronic wasting disease in mule and white-tailed deer by RT-QuIC analysis of outer ear.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

OPPORTUNISTIC SURVEILLANCE OF CAPTIVE AND FREE-RANGING BIGHORN SHEEP (OVIS CANADENSIS) IN COLORADO, USA, FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES.

Bighorn sheep (Ovis canadensis) are predicted to have a degree of susceptibility to the transmissible spongiform encephalopathies (TSE) chronic wasting disease and scrapie. We opportunistically screened 127 captive bighorn sheep and 152 free-ranging bighorn sheep in Colorado, US for the presence of TSE over a period of 35 yr. None of the animals demonstrated clinical signs, gross pathology, histopathology, or immunohistochemical staining patterns suggestive of TSE.

Tissue Residue Levels of the Tranquilizer Combination of Butorphanol, Azaperone, and Medetomidine, and the Antagonists, Naltrexone, Atipamezole, and Tolazoline, in Black Bears (Ursus americanus) Postimmobilization.

The tranquilizer combination of butorphanol, azaperone, and medetomidine (BAM) has shown good efficacy for immobilization of wildlife, including black bears (Ursus americanus). BAM is antagonized with a combination of naltrexone and atipamezole. We immobilized 19 adult captive wild caught black bears and, except for three bears that were euthanized immediately, bears were recovered with naltrexone and atipamezole. Tissue residues (≥0.01 ppm) for the tranquilizers butorphanol, azaperone, and medetomidine were detected in liver and muscle of all three bears euthanized on day 0 postinjection (PI). Azaperone was not detected after 1 d PI. Residue for medetomidine was detected in two bears: in the liver 3 d PI and in the kidney 6 d PI. Butorphanol was reported in three bears: in fat 5 d PI, in kidney 6 d PI, and, surprisingly, in kidney, muscle, and fat 7 d PI. No tissue residues were detected in the three bears euthanized at 8 d PI. Tissue residues for the antagonists, naltrexone and atipamezole, were detected in bears euthanized 2 and 6 d PI, but not in tissues from animals euthanized at 7 or 8 d PI.

EFFECT OF ORAL COPPER SUPPLEMENTATION ON SUSCEPTIBILITY IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) TO CHRONIC WASTING DISEASE.

Chronic wasting disease (CWD) is an infectious disease, but reported associations suggest several metals-especially copper (Cu) and manganese-potentially play a role in this and other prion diseases. To assess the utility of dietary Cu supplementation in protecting white-tailed deer (Odocoileus virginianus) from CWD, we compared incidence and disease course among individuals naturally exposed to CWD while being maintained on sustained-release Cu boluses or unsupplemented (control). Oral Cu supplementation increased liver tissue Cu concentrations compared to controls but did not affect susceptibility to CWD or survival after natural exposure in the captive white-tailed deer we studied. Over the 27 mo study, 89% (8/9) of the Cu-supplemented deer and 86% (6/7) of control deer became CWD-infected. Survival to 27 mo postexposure did not differ between Cu-supplemented and control deer: model-averaged survival probabilities to 27 mo were 0.45-0.47 for all combinations of Cu treatment and PRNP gene haplotype presence. The PRNP gene haplotype influenced the probability of deer remaining biopsy negative for at least 17 mo but did not affect overall susceptibility.

HUNTING PRESSURE MODULATES PRION INFECTION RISK IN MULE DEER HERDS.

The emergence of chronic wasting disease, an infectious prion disease of multiple deer species, has motivated international calls for sustainable, socially accepted control measures. Here, we describe long-term, spatially replicated relationships in Colorado, US, mule deer (Odocoileus hemionus) herds that show hunting pressure can modulate apparent epidemic dynamics as reflected by prevalence trends. Across 12 areas in Colorado studied between 2002-18, those with the largest declines in annual hunting license numbers (pressure) showed the largest increases in the proportion of infected adult (≥2-yr-old) male deer killed by hunters (prevalence); prevalence trends were comparatively flat in most areas where license numbers had been maintained or increased. The mean number of licenses issued in the 2 yr prior best explained observed patterns: increasing licenses lowered subsequent risk of harvesting an infected deer, and decreasing licenses increased that risk. Our findings suggest that harvesting mule deer with sufficient hunting pressure might control chronic wasting disease-especially when prevalence is low-but that harvest prescriptions promoting an abundance of mature male deer contribute to the exponential growth of epidemics.

Episodic Prenatal Exposure To Ethanol Affects Postnatal Neurogenesis In The Macaque Dentate Gyrus And Visual Recognition Memory.

Fetal alcohol syndrome (FAS) is a prime cause of cognitive dysfunction. The present study tested the hypotheses (a) that gestational ethanol exposure results in deficits in hippocampal-related behaviors and associated neurogenesis and (b) that the period of gastrulation is a time of vulnerability. Pregnant macaques were intubated with ethanol or saline once per week for 3, 6, or 24 weeks. Exposures included or omitted the period of gastrulation. Offspring were given behavioral tests including a Visual-Paired Comparison (VPC), a hippocampal-associated memory task, and euthanized as adolescents. Their dentate gyri were processed for immunohistochemical identification of cells passing through the cell cycle (Ki-67 and proliferating cell nuclear antigen), exiting the cell cycle (p21), or passing through early stages of neuronal morphogenesis (Tuj1). Performance in neurobehavioral tasks was unaffected by ethanol exposure, the notable exception being performance in the VPC that was poorer for macaques exposed to ethanol including gastrulation. Anatomical studies show that the expression of Ki-67 was greater and ratio of p21-positive cells to the ratio of Ki-67-expressing cells was lower in animals in which the ethanol exposure included gastrulation. On the other hand, no ethanol-induced differences in TuJ1 expression were detected. Thus, the dentate gyrus is a bellwether of long-term consequences of gestational ethanol exposure. Targeted effects of ethanol on early neural generation (cell cycle and cycle exit) correlate with the timing-dependent degradation in VPC performance and exposure during gastrulation results in notable deficits. These changes evidence a pattern of fetal programming underlying FAS.

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons.

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer's seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106 Hofmeister analysis also improved detection of sporadic Creutzfeldt-Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases.

Oral anticoagulants usage in Japanese patients aged 18-74 years with non-valvular atrial fibrillation: a retrospective analysis based on insurance claims data.

BACKGROUND: Oral anticoagulants use has increased rapidly, internationally. Here we look at risks and benefits, based on Japanese data, of therapy with low risk non-valvular atrial fibrillation patients. OBJECTIVES: Using a health insurance claims data set we assessed: (i) oral anticoagulants usage in Japan, and (ii) efficacy and safety of dabigatran compared with warfarin, in Japanese patients with non-valvular atrial fibrillation, aged 18-74 years. METHODS: We identified 4380 non-valvular atrial fibrillation patients treated with anticoagulants between 1 January 2005, and 28 February 2014, and estimated the adjusted hazard ratio for stroke or systemic embolism, and any hemorrhagic event (Cox proportional hazards regression model with stabilized inverse probability treatment weighting). RESULTS: The data included 101 989 anticoagulant prescriptions for 4380 patients, of which direct oral anticoagulants increased to 40.0% of the total by the end of the study. After applying exclusion criteria, 1536 new non-valvular atrial fibrillation patients were identified, including 1071 treated with warfarin and 465 with dabigatran. Mean ages were 56.11 ± 9.70 years for warfarin, and 55.80 ± 9.65 years for dabigatran. The adjusted hazard ratio (95% confidence interval), comparing dabigatran with warfarin, was 0.48 (0.25-0.91) for stroke or systemic embolism, and 0.91 (0.60-1.39) for any hemorrhage including intracranial and gastrointestinal. CONCLUSIONS: Number of patients prescribed direct oral anticoagulants steadily increased, and incidence of all-cause bleeding related to dabigatran was similar to warfarin, in our study population of younger non-valvular atrial fibrillation patients. Dabigatran, compared with warfarin, generally reduced risk of all-cause stroke and systemic embolism.

History of Amusia.

We live in a world surrounded by sound. Throughout life, we are exposed to music: from lullabies and songs taught at school to instrumental music both heard and played for pleasure. Every nation, along with its own language, has unique forms of music and dance. "Music knows no boundaries," as the saying goes. Just as language impairment is known as "aphasia," impairment of the perception of music is called "amusia." In this article, we will first classify the types of amusia. This will be followed by an introduction to the classical research of Salomon Eberhard Henschen (1847-1930), and to a discussion of higher auditory functions in which we highlight cases of amusia encountered in a person and through the literature.

Ethanol-induced DNA repair in neural stem cells is transforming growth factor ß1-dependent.

Following neurotoxic damage, cells repair their DNA, and survive or undergo apoptosis. This study tests the hypothesis that ethanol induces a DNA damage response (DDR) in neural stem cells (NSCs) that promotes excision repair (ER) and this repair is influenced by the growth factor environment. Non-immortalized NSCs treated with fibroblast growth factor 2 or transforming growth factor (TGF) ß1 were exposed to ethanol. Ethanol increased total DNA damage, reactive oxygen species, and oxidized DNA bases. TGFß1 potentiated these toxic effects. Transcriptional analyses of cultured NSCs revealed ethanol-induced increases in transcripts related to the DDR (e.g., Hus1 and p53), base ER (e.g., Mutyh and Nthl1), and nucleotide ER (e.g., Xpc), particularly in the presence of TGFß1. Expression and activity of ER proteins were affected by ethanol. Similar changes occurred in proliferating cells of ethanol-treated mouse fetuses. Ethanol-induced DNA repair in NSCs depends on the ambient growth factors. Gene products for DNA repair in stem cells are among the first biomarkers identifying fetal alcohol-induced damage.

p53-Mediated Activities in NS-5 Neural Stem Cells: Effects of Ethanol.

BACKGROUND: Transforming growth factor (TGF) ß1 and ethanol (EtOH) powerfully inhibit the proliferation, DNA repair, and survival of neural stem cells (NSCs). The present study tests the hypothesis that the EtOH-induced DNA damage response is mediated through p53 pathways and influenced by growth factor signals. METHODS: Cultures of nonimmortalized NSCs, NS-5 cells, were transfected with p53 siRNA, exposed to either the mitogenic fibroblast growth factor (FGF) 2 or antimitogenic TGFß1, and to EtOH. Stage-specific cellular and genomic responses were examined. RESULTS: p53 status, EtOH exposure, and growth factor significantly affected the expression of transcripts related to the DNA damage response (including those coding for excision repair proteins), mitotic promoters, and regulators of cell death via the tumor necrosis factor pathway. There were significant compensatory increases in p53 family members, p63 and p73, notably in regard to the regulation of cell cycle restriction and apoptosis. Treatment with p53 siRNA potentiated EtOH- and TGFß1-induced changes in the numbers of proliferating NSCs and increased the proportion of NSCs expressing the apoptotic marker annexin V. CONCLUSIONS: Thus, it appears that EtOH and TGFß1 affect proliferation, DNA repair, and survival of NSCs via p53-mediated activities.