The differential proteomic response to ischemic stroke in appalachian subjects treated with mechanical thrombectomy.

INTRODUCTION: The Appalachia region of North America is known to have significant health disparities, specifically, worse risk factors and outcomes for stroke. Appalachians are more likely to have comorbidities related to stroke, such as diabetes, obesity, and tobacco use, and are often less likely to have stroke interventions, such as mechanical thrombectomy (MT), for emergent large vessel occlusion (ELVO). As our Comprehensive Stroke Center directly serves stroke subjects from both Appalachian and non-Appalachian areas, inflammatory proteomic biomarkers were identified associated with stroke outcomes specific to subjects residing in Appalachia. METHODS: There were 81 subjects that met inclusion criteria for this study. These subjects underwent MT for ELVO, and carotid arterial blood samples acquired at time of intervention were sent for proteomic analysis. Samples were processed in accordance with the Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC; clinicaltrials.gov; NCT03153683). Statistical analyses were utilized to examine whether relationships between protein expression and outcomes differed by Appalachian status for functional (NIH Stroke Scale; NIHSS and Modified Rankin Score; mRS), and cognitive outcomes (Montreal Cognitive Assessment; MoCA). RESULTS: No significant differences were found in demographic data or co-morbidities when comparing Appalachian to non-Appalachian subjects. However, time from stroke onset to treatment (last known normal) was significantly longer and edema volume significantly higher in patients from Appalachia. Further, when comparing Appalachian to non-Appalachian subjects, there were significant unadjusted differences in the NIHSS functional outcome. A comprehensive analysis of 184 proteins from Olink proteomic (92 Cardiometabolic and 92 Inflammation panels) showed that the association between protein expression outcomes significantly differed by Appalachian status for seven proteins for the NIHSS, two proteins for the MoCA, and three for the mRS. CONCLUSION: Our study utilizes an ELVO tissue bank and registry to investigate the intracranial/intravascular proteomic environment occurring at the time of thrombectomy. We found that patients presenting from Appalachian areas have different levels of proteomic expression at the time of MT when compared to patients presenting from non-Appalachian areas. These proteins differentially relate to stroke outcome and could be used as prognostic biomarkers, or as targets for novel therapies. The identification of a disparate proteomic response in Appalachian patients provides initial insight to the biological basis for health disparity. Nevertheless, further investigations through community-based studies are imperative to elucidate the underlying causes of this differential response.

Regenerating Family Member 3 Alpha Is Predictive of Mortality Following Emergent Large Vessel Occlusion.

Regenerating Family Member 3 Alpha (REG3A) is an antimicrobial protein secreted by the intestine and pancreas with additional immunomodulatory properties. Previously, we published that REG3A expression in ischemic stroke patient systemic blood, during mechanical thrombectomy (MT), is significantly associated with inflammatory cytokines and patient function on admission. This paper, however, did not investigate post-acute death rates. Therefore, we investigated plasma REG3A protein expression, during MT, in patients (n = 141) that survived or died within the end of the follow-up after MT. Subjects who died had significantly higher systemic plasma REG3A levels at the time of MT compared to survivors (p = 0.001). Age, sex, time from last known normal, and admission NIHSS were included as predictors to control for confounding variables and were all examined to determine their association in patient mortality. Logistic regression was used to demonstrate that higher odds of death were associated with increased REG3A levels (p = 0.002). REG3A demonstrated acceptable discrimination (AUC (95% CI): 0.669 (0.566-0.772) in predicting mortality. The overall model with age, sex, time from last known normal, and admission NIHSS discriminated well between survivors and those who died (AUC (95% CI): 0.784 (0.703-0.864)). In conclusion, REG3A could be promising as a biomarker to prognosticate stroke outcomes and stratify high-risk groups following acute ischemic stroke.

Inflammatory-associated proteomic predictors of cognitive outcome in subjects with ELVO treated by mechanical thrombectomy.

BACKGROUND: Emergent Large Vessel Occlusion (ELVO) stroke causes devastating vascular events which can lead to significant cognitive decline and dementia. In the subset of ELVO subjects treated with mechanical thrombectomy (MT) at our institution, we aimed to identify systemic and intracranial proteins predictive of cognitive function at time of discharge and at 90-days. These proteomic biomarkers may serve as prognostic indicators of recovery, as well as potential targets for novel/existing therapeutics to be delivered during the subacute stage of stroke recovery. METHODS: At the University of Kentucky Center for Advanced Translational Stroke Sciences, the BACTRAC tissue registry (clinicaltrials.gov; NCT03153683) of human biospecimens acquired during ELVO stroke by MT is utilized for research. Clinical data are collected on each enrolled subject who meets inclusion criteria. Blood samples obtained during thrombectomy were sent to Olink Proteomics for proteomic expression values. Montreal Cognitive Assessments (MoCA) were evaluated with categorical variables using ANOVA and t-tests, and continuous variables using Pearson correlations. RESULTS: There were n = 52 subjects with discharge MoCA scores and n = 28 subjects with 90-day MoCA scores. Several systemic and intracranial proteins were identified as having significant correlations to discharge MoCA scores as well as 90-day MoCA scores. Highlighted proteins included s-DPP4, CCL11, IGFBP3, DNER, NRP1, MCP1, and COMP. CONCLUSION: We set out to identify proteomic predictors and potential therapeutic targets related to cognitive outcomes in ELVO subjects undergoing MT. Here, we identify several proteins which predicted MoCA after MT, which may serve as therapeutic targets to lessen post-stroke cognitive decline.

Antimicrobial protein REG3A and signaling networks are predictive of stroke outcomes.

Regenerating Family Member 3 Alpha (REG3A) is a multifunctional protein with antimicrobial activity, and primarily secreted by the intestine and pancreas. Studies have shown an increased expression of REG3A in systemic inflammatory responses to acute injury and infection, but studies investigating REG3A during the pathogenesis of ischemic stroke are limited. The aims of this study were to examine the associations between arterial expression of REG3A and other arterial inflammatory proteins implicated in stroke pathogenesis, as well as associations between REG3A and markers of poor outcome for ischemic stroke. The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate intracranial arterial blood (i.e. distal to thrombus) and systemic arterial blood (i.e. carotid). Samples were analyzed by Olink Proteomics for N = 42 subjects. Statistical analyses of plasma proteins included 2-sample t-tests, spearman and biserial correlations, and robust regression models to elucidate network signaling and association to clinical outcomes. Results indicated that levels of systemic REG3A were positively correlated with inflammatory proteins interleukin IL6 (R = 0.344, p = 0.030) and IL17C (R = 0.468, p = 0.002). 2-sided t- tests examining differences of systemic REG3A within quartiles of NIHSS admission score depicted significant differences between quartiles. Those with NIHSS scores corresponding to moderate and moderate-severe neurofunctional deficits had significantly higher levels of systemic REG3A compared to those with NIHSS scores corresponding to mild and mild-moderate neurofunctional deficits (p = 0.016). STRING analyses of proteins in each robust regression model demonstrated substantial networking between REG3A and other systemic proteins highly relevant to ischemic stroke. The present study provides novel data on systemic REG3A in the context of ischemic stroke. These results demonstrate the influential role of REG3A regarding surrogate functional and radiographic outcomes of stroke severity. Additionally, they provide novel insight into the role of REG3A and related proteins during the complex neuroinflammatory process of ischemic stroke. These data provide a foundation for future studies to investigate REG3A and related networking proteins as potential biomarkers with prognostic potential, as well as potential therapeutic targets.

MANF is neuroprotective against ethanol-induced neurodegeneration through ameliorating ER stress.

Fetal alcohol spectrum disorders (FASD) are a spectrum of developmental disorders caused by prenatal alcohol exposure. Neuronal loss or neurodegeneration in the central nervous system (CNS) is one of the most devastating features in FASD. It is imperative to delineate the underlying mechanisms to facilitate the treatment of FASD. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases, including ethanol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in response to ethanol exposure during the brain development. To investigate the role of MANF in ethanol-induced neurodegeneration and its association with ER stress regulation, we established a CNS-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency exacerbated ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, using an animal model of ER-stress-induced neurodegeneration, we demonstrated that MANF deficiency potentiated tunicamycin (TM)-induced ER stress and neurodegeneration. A whole transcriptome RNA sequencing also supported the functionality of MANF in ER stress modulation and revealed targets that may mediate the ER stress-buffering capacity of MANF. Collectively, these results suggest that MANF is a neurotrophic factor that can protect neurons against ethanol-induced neurodegeneration by ameliorating ER stress.

Intracranial VCAM1 at time of mechanical thrombectomy predicts ischemic stroke severity.

BACKGROUND: Emergent large vessel occlusion (ELVO) strokes are devastating ischemic vascular events for which novel treatment options are needed. Using vascular cell adhesion molecule 1 (VCAM1) as a prototype, the objective of this study was to identify proteomic biomarkers and network signaling functions that are potential therapeutic targets for adjuvant treatment for mechanical thrombectomy. METHODS: The blood and clot thrombectomy and collaboration (BACTRAC) study is a continually enrolling tissue bank and registry from stroke patients undergoing mechanical thrombectomy. Plasma proteins from intracranial (distal to clot) and systemic arterial blood (carotid) were analyzed by Olink Proteomics for N=42 subjects. Statistical analysis of plasma proteomics used independent sample t tests, correlations, linear regression, and robust regression models to determine network signaling and predictors of clinical outcomes. Data and network analyses were performed using IBM SPSS Statistics, SAS v 9.4, and STRING V11. RESULTS: Increased systemic (p<0.001) and intracranial (p=0.013) levels of VCAM1 were associated with the presence of hypertension. Intracranial VCAM1 was positively correlated to both infarct volume (p=0.032; r=0.34) and edema volume (p=0.026; r=0.35). The %∆ in NIHSS from admittance to discharge was found to be significantly correlated to both systemic (p=0.013; r = -0.409) and intracranial (p=0.011; r = -0.421) VCAM1 levels indicating elevated levels of systemic and intracranial VCAM1 are associated with reduced improvement of stroke severity based on NIHSS from admittance to discharge. STRING-generated analyses identified biologic functional descriptions as well as function-associated proteins from the predictive models of infarct and edema volume. CONCLUSIONS: The current study provides novel data on systemic and intracranial VCAM1 in relation to stroke comorbidities, stroke severity, functional outcomes, and the role VCAM1 plays in complex protein-protein signaling pathways. These data will allow future studies to develop predictive biomarkers and proteomic targets for drug development to improve our ability to treat a devastating pathology.

Early Ethanol Exposure Inhibits the Differentiation of Hippocampal Dentate Gyrus Granule Cells in a Mouse Model of Fetal Alcohol Spectrum Disorders.

BACKGROUND: Alcohol consumption during pregnancy may damage the developing central nervous system of the fetus and lead to brain structural and functional deficits in the children, known as fetal alcohol spectrum disorders. The underlying mechanisms have not been fully elucidated. Previously, using a third trimester-equivalent mouse model, ethanol (EtOH)-induced behavioral deficits (including spatial learning and memory dysfunction) in the mice were detected on postnatal day (PD) 35. The hippocampal formation is critically involved in spatial learning/memory and contains 2 major neuron populations: the pyramidal cells in the hippocampus proper and the dentate gyrus granule cells (DGGCs) in the dentate gyrus (DG). In rodents, while the pyramidal cells are almost exclusively generated prenatally, the DG granule neurons are majorly generated during the first 2 weeks postnatally, which coincides with the period of EtOH exposure in our mouse model. Therefore, in the current study the effects of EtOH exposure on the development of the DGGCs were examined. METHODS: C57BL/6 mice were treated with 4 g/kg of EtOH by intubation on PD 4 to 10 to mimic alcohol exposure to the fetus during the third trimester in humans, and the development of DGGCs was examined by immunohistochemistry and quantified on PD 35. RESULTS: EtOH exposure does not affect the number of total or newly generated DGGCs, but reduces the number of mature DGGCs on PD 35 in both male and female mice. The ratio of immature DGGCs over total DGGCs was increased, and the ratio of mature DGGCs over total DGGCs was decreased by EtOH exposure. In addition, no sex-dependent effects of EtOH treatment were detected. CONCLUSION: Our data indicate that EtOH exposure in mice during PD 4 to 10 does not affect the generation/proliferation but inhibits the differentiation of the DGGCs on PD 35.

4-Phenylbutyric Acid Protects Against Ethanol-Induced Damage in the Developing Mouse Brain.

BACKGROUND: Ethanol (EtOH) exposure during pregnancy may result in fetal alcohol spectrum disorders (FASD). One of the most deleterious consequences of EtOH exposure is neuronal loss in the developing brain. Previously, we showed that EtOH exposure induced neuroapoptosis in the brain of postnatal day 4 (PD4) mice but not PD12 mice. This differential susceptibility may result from an insufficient cellular stress response system such as unfolded protein response (also known as endoplasmic reticulum [ER] stress) in PD4 mice. In this study, we compared the effect of EtOH on ER stress in PD4 and PD12 mice and determined whether the inhibition of ER stress could protect the developing brain against EtOH damage. METHODS: We used a third-trimester equivalent mouse model of FASD. PD4 and PD12 C57BL/6 mice were subcutaneously injected with saline (control), EtOH, EtOH plus 4-phenylbutyric acid (4-PBA), a chemical chaperone known as ER stress inhibitor, and 4-PBA alone. The expression of apoptosis marker, ER stress markers, and markers for glial cell activation was examined in the cerebral cortex. RESULTS: EtOH induced neuroapoptosis and increased the expression of ER stress markers, such as activating transcription factor 6, 78-kDa glucose-regulated protein, inositol-requiring enzyme 1α, mesencephalic astrocyte-derived neurotrophic factor, and caspase-12 in PD4 but not PD12 mice. EtOH exposure also activated microglia and astrocytes. Interestingly, treatment with 4-PBA attenuated EtOH-induced neuroapoptosis. Moreover, 4-PBA inhibited the expression of the aforementioned ER stress markers and EtOH-induced glial activation in PD4 mice. CONCLUSIONS: ER stress plays an important role in EtOH-induced damage to the developing brain. Inhibition of ER stress is neuroprotective and may provide a new therapeutic strategy for treating FASD.

Role of MCP-1 and CCR2 in ethanol-induced neuroinflammation and neurodegeneration in the developing brain.

BACKGROUND: Neuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD). Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. FASD is the leading cause of mental retardation, and one of the most devastating outcomes of FASD is the loss of neurons in the central nervous system (CNS). The underlying molecular mechanisms, however, remain unclear. We hypothesize that MCP-1/CCR2 signaling mediates ethanol-induced neuroinflammation and microglial activation, which exacerbates neurodegeneration in the developing brain. METHODS: C57BL/6 mice and mice deficient of MCP-1 (MCP-1-/-) and CCR2 (CCR2-/-) were exposed to ethanol on postnatal day 4 (PD4). Neuroinflammation, and microglial activation, and neurodegeneration in the brain were evaluated by immunohistochemistry and immunoblotting. A neuronal and microglial co-culture system was used to evaluate the role of microglia and MCP-1/CCR2 signaling in ethanol-induced neurodegeneration. Specific inhibitors were employed to delineate the involved signaling pathways. RESULTS: Ethanol-induced microglial activation, neuroinflammation, and a drastic increase in the mRNA and protein levels of MCP-1. Treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. MCP-1-/- and CCR2-/- mice were more resistant to ethanol-induced neuroapoptosis. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3ß, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3ß. CONCLUSION: MCP-1/CCR2 signaling played an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain. The effects may be mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3ß.

Binge ethanol exposure induces endoplasmic reticulum stress in the brain of adult mice.

Alcohol abuse causes brain damage and cognitive dysfunction. However, the underlying mechanisms remain elusive. Endoplasmic reticulum (ER) acts as machinery to ensure the proper folding of newly synthesized proteins. The perturbation of ER, i.e., ER stress, plays a pivotal role in some neurological disorders. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is involved in the regulation of ER stress. The current study sought to determine whether binge ethanol exposure induces ER stress in adult mouse brain and the role mTOR signaling during this process. Adult C57BL6 mice received binge ethanol exposure by daily gavage (5 g/kg, 25% ethanol w/v) for 1, 5 or 10 days. Binge ethanol exposure caused neurodegeneration and neuroinflammation after 5 days of exposure, and a concomitant increase of ER stress and inhibition of mTOR. However, ethanol exposure did not significantly alter spatial learning and memory, and spontaneous locomotor activity. Ethanol treatment induced ER stress and the death of cultured neuronal cells. Cotreatment with an ER stress inhibitor, sodium 4-phenylbutyrate (4-PBA) significantly diminished ethanol-induced ER stress and neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration. Furthermore, the blockage of mTOR activity by rapamycin increased ER stress in cultured neuronal cells; whereas the activation or inhibition of ER stress by tunicamycin or 4-PBA respectively had little effects on mTOR signaling. These results suggested that mTOR signaling is upstream of ER stress and may thereby mediate ethanol-induced ER stress.

Neonatal Ethanol Exposure Causes Behavioral Deficits in Young Mice.

BACKGROUND: Fetal ethanol (EtOH) exposure can damage the developing central nervous system and lead to cognitive and behavioral deficits, known as fetal alcohol spectrum disorders (FASD). EtOH exposure to mouse pups during early neonatal development was used as a model of EtOH exposure that overlaps the human third-trimester "brain growth spurt"-a model that has been widely used to study FASD in rats. METHODS: C57BL/6 male and female mice were exposed to EtOH (4 g/kg/d) on postnatal days (PD) 4 to 10 by oral intubation. Intubated and nontreated controls were also included. Behavioral testing of the offspring, including open field, elevated plus maze, and Morris water maze, was performed on PD 20 to 45. RESULTS: EtOH exposure during PD 4 to 10 resulted in hyperactivity and deficits in learning and memory in young mice with no apparent sex differences. CONCLUSIONS: Based on these data, this neonatal intubation mouse model may be useful for future mechanistic and genetic studies of FASD and for screening of novel therapeutic agents.

Ethanol-induced damage to the developing spinal cord: The involvement of CCR2 signaling.

Ethanol exposure during development causes fetal alcohol spectrum disorders (FASD). A large body of evidence shows that ethanol produces multiple abnormalities in the developing central nervous system (CNS), such as smaller brain size, reduced volume of cerebral white matter, permanent loss of neurons, and alterations in synaptogenesis and myelinogenesis. The effects of ethanol on the developing spinal cord, however, receive little attention and remain unclear. We used a third trimester equivalent mouse model to investigate the effect of ethanol on the developing spinal cord. Ethanol caused apoptosis and neurodegeneration in the dorsal horn neurons of mice of early postnatal days, which was accompanied by glial activation, macrophage infiltration, and increased expression of CCR2, a receptor for monocyte chemoattractant protein 1 (MCP-1). Ethanol-induced neuronal death during development resulted in permanent loss of spinal cord neurons in adult mice. Ethanol stimulated endoplasmic reticulum (ER) stress and oxidative stress, and activated glycogen synthase kinase 3ß (GSK3ß) and c-Jun N-terminal kinase (JNK) pathways. Knocking out MCP-1 or CCR2 made mice resistant to ethanol-induced apoptosis, ER stress, glial activation, and activation of GSK3ß and JNK. CCR2 knock out offered much better protection against ethanol-induced damage to the spinal cord. Thus, developmental ethanol exposure caused permanent loss of spinal cord neurons and CCR2 signaling played an important role in ethanol neurotoxicity.

Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells.

Thiamine (vitamin B1) deficiency (TD) plays a major role in the etiology of Wernicke's encephalopathy (WE) which is a severe neurological disorder. TD induces selective neuronal cell death, neuroinflammation, endoplasmic reticulum (ER) stress and oxidative stress in the brain which are commonly observed in many aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and progressive supranuclear palsy (PSP). However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research is hindered due to the lack of appropriate in vitro models. The neurons derived for the human induced pluripotent stem cells (hiPSCs) provide a relevant and powerful tool for the research in pharmaceutical and environmental neurotoxicity. In this study, we for the first time used human induced pluripotent stem cells (hiPSCs)-derived neurons (iCell neurons) to investigate the mechanisms of TD-induced neurodegeneration. We showed that TD caused a concentration- and duration-dependent death of iCell neurons. TD induced ER stress which was evident by the increase in ER stress markers, such as GRP78, XBP-1, CHOP, ATF-6, phosphorylated eIF2α, and cleaved caspase-12. TD also triggered oxidative stress which was shown by the increase in the expression 2,4-dinitrophenyl (DNP) and 4-hydroxynonenal (HNE). ER stress inhibitors (STF-083010 and salubrinal) and antioxidant N-acetyl cysteine (NAC) were effective in alleviating TD-induced death of iCell neurons, supporting the involvement of ER stress and oxidative stress. It establishes that the iCell neurons are a novel tool to investigate cellular and molecular mechanisms for TD-induced neurodegeneration.

The accuracy of objective threshold determination at low frequencies: comparison of different auditory brainstem response (ABR) and auditory steady state response (ASSR) methods.

OBJECTIVE: The hearing threshold at 500 Hz was estimated using five methods which are suitable for the low frequency range: Low-Chirp BERA (LCBERA), Notched-noise BERA (NNBERA), Narrow band CE-Chirp BERA (NBCBERA) and Narrow band CE-Chirp ASSR (NBCASSR) (40/90 Hz). The slope of the discrimination function of each method was used for determination of the most efficient method. The threshold values were compared and the corresponding odds ratios (OR) were calculated. DESIGN: All methods were applied to each subject. Stimulus levels were arranged individually. Response detection was carried out by visual inspection of the records in case of BERA and automatically in case of ASSR. Each individual series of recordings was converted to a dichotomous function indicating whether or not a response was discernible and a continuous method-specific discrimination function was constructed. This function was realised by a Boltzmann function whose slope in the inflection point serves as quality measure. Additionally, an OR evaluation was carried out in order to validate the significance of results. STUDY SAMPLE: Twenty five normal hearing adults (aged 18-30 years) were tested. RESULTS: LCBERA proved to have the highest reliability according to the slope of the Boltzmann function, the comparison of threshold values and OR. CONCLUSIONS: The LCBERA is recommended for use in routine clinical practice.

ErbB2 and p38γ MAPK mediate alcohol-induced increase in breast cancer stem cells and metastasis.

BACKGROUND: Both epidemiological and experimental studies suggest that excessive alcohol exposure increases the risk for breast cancer and enhances metastasis/recurrence. We have previously demonstrated that alcohol enhanced the migration/invasion of breast cancer cells and cancer cells overexpressing ErbB2/HER2 were more sensitive to alcohol exposure. However, the underlying mechanisms remain unclear. This study was designed to investigate the mechanisms underlying alcohol-enhanced aggressiveness of breast cancer. Cancer stem cells (CSCs) play a critical role in cancer metastasis and recurrence. METHODS: We evaluated the effect of chronic alcohol exposure on mammary tumor development/metastasis in MMTV-neu transgenic mice and investigated the cell signaling in response to alcohol exposure in breast cancer cells overexpressing ErbB2/HER2. RESULTS AND DISCUSSION: Chronic alcohol exposure increased breast cancer stem cell-like CSC population and enhanced the lung and colon metastasis in MMTV-neu transgenic mice. Alcohol exposure caused a drastic increase in CSC population and mammosphere formation in breast cancer cells overexpressing ErbB2/HER2. Alcohol exposure stimulated the phosphorylation of p38γ MAPK (p-p38γ) which was co-localized with phosphorylated ErbB2 and CSCs in the mammary tumor tissues. In vitro results confirmed that alcohol activated ErbB2/HER2 and selectively increased p-p38γ MAPK as well as the interaction between p38γ MAPK and its substrate, SAP97. However, alcohol did not affect the expression/phosphorylation of p38α/ß MAPKs. In breast cancer cell lines, high expression of ErbB2 and p-p38γ MAPK was generally correlated with more CSC population. Blocking ErbB2 signaling abolished heregulin ß1- and alcohol-stimulated p-p38γ MAPK and its association with SAP97. More importantly, p38γ MAPK siRNA significantly inhibited an alcohol-induced increase in CSC population, mammosphere formation and migration/invasion of breast cancer cells overexpressing ErbB2. CONCLUSIONS: p38γ MAPK is downstream of ErbB2 and plays an important role in alcohol-enhanced aggressiveness of breast cancer. Therefore, in addition to ErbB2/HER2, p38γ MAPK may be a potential target for the treatment of alcohol-enhanced cancer aggressiveness.

Role of MCP-1 in alcohol-induced aggressiveness of colorectal cancer cells.

Epidemiological studies demonstrate that alcohol consumption is associated with an increased risk of colorectal cancer (CRC). In addition to promoting carcinogenesis, alcohol may also accelerate the progression of existing CRC. We hypothesized that alcohol may enhance the aggressiveness of CRC. In this study, we investigated the effect of alcohol on the migration/invasion and metastasis of CRC. Alcohol increased the migration/invasion of colorectal cancer cells (DLD1, HCT116, HT29, and SW480) in a concentration-dependent manner. Among these colon cancer cell lines, HCT116 cells were most responsive while HT29 cells were the least responsive to ethanol-stimulated cell migration/invasion. These in vitro results were supported by animal studies which demonstrated that ethanol enhanced the metastasis of colorectal cancer cells to the liver and lung. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in regulating tumor microenvironment and metastasis. Alcohol increased the expression of MCP-1 and its receptor CCR2 at both protein and mRNA levels. The pattern of alcohol-induced alterations in MCP-1 expression was consistent with its effect on migration/invasion; HCT116 cells displayed the highest up-regulation of MCP-1/CCR2 in response to alcohol exposure. An antagonist of CCR2 blocked alcohol-stimulated migration. Alcohol caused an initial cytosolic accumulation of ß-catenin and its subsequent nuclear translocation by inhibiting GSK3ß activity. Alcohol stimulated the activity of MCP-1 gene promoter in a ß-catenin-dependent manner. Furthermore, knock-down of MCP-1/CCR2 or ß-catenin was sufficient to inhibit alcohol-induced cell migration/invasion. Together, these results suggested that alcohol may promote the metastasis of CRC through modulating GSK3ß/ß-catenin/MCP-1 pathway.

Chronic plus binge ethanol exposure causes more severe pancreatic injury and inflammation.

Alcohol abuse increases the risk for pancreatitis. The pattern of alcohol drinking may impact its effect. We tested a hypothesis that chronic ethanol consumption in combination with binge exposure imposes more severe damage to the pancreas. C57BL/6 mice were divided into four groups: control, chronic ethanol exposure, binge ethanol exposure and chronic plus binge ethanol exposure. For the control group, mice were fed with a liquid diet for two weeks. For the chronic ethanol exposure group, mice were fed with a liquid diet containing 5% ethanol for two weeks. In the binge ethanol exposure group, mice were treated with ethanol by gavage (5g/kg, 25% ethanol w/v) daily for 3days. For the chronic plus binge exposure group, mice were fed with a liquid diet containing 5% ethanol for two weeks and exposed to ethanol by gavage during the last 3days. Chronic and binge exposure alone caused minimal pancreatic injury. However, chronic plus binge ethanol exposure induced significant apoptotic cell death. Chronic plus binge ethanol exposure altered the levels of alpha-amylase, glucose and insulin. Chronic plus binge ethanol exposure caused pancreatic inflammation which was shown by the macrophages infiltration and the increase of cytokines and chemokines. Chronic plus binge ethanol exposure increased the expression of ADH1 and CYP2E1. It also induced endoplasmic reticulum stress which was demonstrated by the unfolded protein response. In addition, chronic plus binge ethanol exposure increased protein oxidation and lipid peroxidation, indicating oxidative stress. Therefore, chronic plus binge ethanol exposure is more detrimental to the pancreas.

MicroRNA-29b regulates ethanol-induced neuronal apoptosis in the developing cerebellum through SP1/RAX/PKR cascade.

Neuronal loss is a prominent etiological factor for fetal alcohol spectrum disorders. The cerebellum is one of the areas in the developing central nervous system that is most sensitive to ethanol, especially during the temporal window of ethanol vulnerability. MicroRNAs are small, non-coding RNAs capable of regulating diverse cellular functions including apoptosis. Ethanol exposure has been shown to interfere with the expression of microRNAs. However, the role of microRNAs in ethanol neurotoxicity is still not clear. In the present study, we identified a particular microRNA, miR-29b, as a novel target of ethanol in the developing cerebellar granule neurons. We discovered that ethanol exposure suppressed miR-29b and induced neuronal apoptosis. Overexpression of miR-29b rendered neurons protection against ethanol-induced apoptosis. Furthermore, our data indicated that miR-29b mediated ethanol neurotoxicity through the SP1/RAX/PKR cascade. More importantly, the expression of miR-29b is developmentally regulated, which may account for, at least partially, the temporal window of ethanol sensitivity in the developing cerebellum.

Tunicamycin-induced unfolded protein response in the developing mouse brain.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1-CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress.

Deficient PKR in RAX/PKR Association Ameliorates Ethanol-Induced Neurotoxicity in the Developing Cerebellum.

Ethanol-induced neuronal loss is closely related to the pathogenesis of fetal alcohol spectrum disorders. The cerebellum is one of the brain areas that are most sensitive to ethanol. The mechanism underlying ethanol neurotoxicity remains unclear. Our previous in vitro studies have shown that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) regulates neuronal apoptosis upon ethanol exposure and ethanol activates PKR through association with its intracellular activator RAX. However, the role of PKR and its interaction with RAX in vivo have not been investigated. In the current study, by utilizing N-PKR-/- mice, C57BL/6J mice with a deficient RAX-binding domain in PKR, we determined the critical role of RAX/PKR association in PKR-regulated ethanol neurotoxicity in the developing cerebellum. Our data indicate that while N-PKR-/- mice have a similar BAC profile as wild-type mice, ethanol induces less brain/body mass reduction as well as cerebellar neuronal loss. In addition, ethanol promotes interleukin-1ß (IL-1ß) secretion, and IL-1ß is a master cytokine regulating inflammatory response. Importantly, ethanol-promoted IL-1ß secretion is inhibited in the developing cerebellum of N-PKR-/- mice. Thus, RAX/PKR interaction and PKR activation regulate ethanol neurotoxicity in the developing cerebellum, which may involve ethanol-induced neuroinflammation. Further, PKR could be a possible target for pharmacological intervention to prevent or treat fetal alcohol spectrum disorder (FASD).