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.

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.

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).

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.

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.

Asymptomatic deer excrete infectious prions in faeces.

Infectious prion diseases-scrapie of sheep and chronic wasting disease (CWD) of several species in the deer family-are transmitted naturally within affected host populations. Although several possible sources of contagion have been identified in excretions and secretions from symptomatic animals, the biological importance of these sources in sustaining epidemics remains unclear. Here we show that asymptomatic CWD-infected mule deer (Odocoileus hemionus) excrete CWD prions in their faeces long before they develop clinical signs of prion disease. Intracerebral inoculation of irradiated deer faeces into transgenic mice overexpressing cervid prion protein (PrP) revealed infectivity in 14 of 15 faecal samples collected from five deer at 7-11 months before the onset of neurological disease. Although prion concentrations in deer faeces were considerably lower than in brain tissue from the same deer collected at the end of the disease, the estimated total infectious dose excreted in faeces by an infected deer over the disease course may approximate the total contained in a brain. Prolonged faecal prion excretion by infected deer provides a plausible natural mechanism that might explain the high incidence and efficient horizontal transmission of CWD within deer herds, as well as prion transmission among other susceptible cervids.

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.

In vivo comparison of chronic wasting disease infectivity from deer with variation at prion protein residue 96.

Chronic wasting disease (CWD) is a prion disease of cervids that causes neurodegeneration and death. Susceptibility to prion infections, including CWD, can be dependent on the amino acid sequence of the host prion protein (PrP). Here, CWD agent obtained from a deer expressing the 96SS genotype, associated with partial resistance to CWD, was used to infect transgenic (tg) mice expressing either 96GG or 96SS deer PrP. Transgenic mice expressing 96GG deer PrP succumbed to this agent, but tg mice expressing 96SS deer PrP did not. Additional studies using inocula from 96GG deer showed no transmission to 96SS PrP mice and delayed disease in 96GS mice. Thus, 96S PrP played an inhibitory role in disease progression in tg mice.

Prion disease detection, PMCA kinetics, and IgG in urine from sheep naturally/experimentally infected with scrapie and deer with preclinical/clinical chronic wasting disease.

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrP(Sc) detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrP(Sc) from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrP(C) to PrP(Sc) was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrP(C) source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrP(Sc) from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.

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.

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.

Amyotrophic lateral sclerosis and language dysfunction: kana, kanji and a prescient report in Japanese by Watanabe (1893).

Although Charcot described amyotrophic lateral sclerosis (ALS) in the 1870s, he did not focus on language. And language problems in ALS with or without dementia were long ignored. A recent report by Caselli et al. [Ann Neurol 1993;33:200-207] is accurately regarded as a major breakthrough in studies of language in ALS. However, we discovered a Japanese account written by Watanabe in 1893 describing paragraphia of an aphasic nature, and this is interesting for two reasons. (1) Watanabe's paper is, we believe, the first report of an aphasia associated with motor neuron disease, and predates other reports by 100 years. (2) It sheds light on the dissociated involvement of the two Japanese writing systems: kana (Japanese simple phonograms) and kanji (Japanese morphograms with complex character derived from Chinese characters). In the aphasia reported by Watanabe, the phonograms are more affected than the morphograms. Thus, Watanabe's clinical observation may predict current theories of the way in which these two writing systems involve different intrahemispheric pathways.

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.

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.

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.

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.

Ethanol affects transforming growth factor beta1-initiated signals: cross-talking pathways in the developing rat cerebral wall.

TGFbeta1 regulates early cortical development by moving young neurons from the proliferative population and promoting their migration. Altered TGFbeta1-regulated signaling can lead to abnormal development underlying microencephaly and migratory defects as in attention deficit hyperactivity disorder, dyslexia, and fetal alcohol spectrum disorder. The present study tested the hypotheses that TGFbeta1 signals through cross-talking Smad2/3 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways and that ethanol simulates these TGFbeta1-initiated signals. Slices generated from the telencephalons of 17-d-old rat fetuses were treated with TGFbeta1 (0 or 10 ng/ml), ethanol (0, 200, 400, or 800 mg/dl), and/or a pharmacological agent [to block phosphorylation of Smad2/3 (SB431542 or LY364947) or ERK1/2 (PD98059 or U0126)]. In some experiments, the proliferative compartment (the ventricular and subventricular zones; S/VZ) and the postproliferative compartment (intermediate zone/cortical plate/marginal zone) were examined separately. Smad2/3 phosphorylation increased following treatment with TGFbeta1, ethanol, and PD98059 (or U0126) plus ethanol. In contrast, SB431542 (and LY364947) blocked Smad2/3 activation and led to the phosphorylation of ERK1/2. These changes revealed cross talk between the two TGFbeta1-responsive pathways. TGFbeta1-induced effects were primarily in the S/VZ, whereas ethanol induced activation in both compartments. In summary, TGFbeta1 activates two separate pathways (Smad2/3 and ERK1/2) that actively interact. Ethanol simulates TGFbeta1-induced changes in these signaling systems. Each pathway is preferentially activated during specific developmental events: the Smad2/3 pathway is key for cells exiting from the cycling population and the ERK1/2 pathway is particularly inducible during neuronal migration.

Ethanol-induced methylation of cell cycle genes in neural stem cells.

Ethanol inhibits the proliferation of neural precursors by altering mitogenic and anti-mitogenic growth factor signaling and can affect global methylation activity in the fetus. We tested the hypothesis that epigenetic modification of specific cell cycle genes underlies the ethanol-induced inhibition of growth factor-regulated cell cycle progression. Monolayer cultures of neural stem cells (NSCs) were treated with fibroblast growth factor 2 or transforming growth factor (TGF) ß1 in the absence or presence of ethanol. Ethanol increased the total length of the cell cycle by elongating the amount of time spent in the gap 1 (G1) and synthesis (S) phases of the cell cycle. Ethanol induced the hypermethylation of multiple cell cycle genes associated with the G1/S and gap 2/mitotic phase (G2/M) checkpoints and increased the expression and activity of DNA methyltransferases. These changes were most pronounced in the presence of TGFß1. Epigenetic alterations paralleled the down-regulation of associated transcripts and other checkpoint-related mRNAs both in vitro (NS-5 cell culture) and in vivo (fetal mouse cortex). Ethanol-induced hypermethylation was accompanied by decreases in the proportion of NSCs expressing associated cell cycle proteins. Thus, ethanol disrupts growth factor-related cell cycle progression by inducing checkpoint restriction at the G1/S transition through a feed-forward system involving the methylation of G2/M regulators.