Sepsis-induced endothelial dysfunction drives acute-on-chronic liver failure through Angiopoietin-2-HGF-C/EBPß pathway.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is an acute liver and multisystem failure in patients with previously stable cirrhosis. A common cause of ACLF is sepsis secondary to bacterial infection. Sepsis-associated ACLF involves a loss of differentiated liver function in the absence of direct liver injury, and its mechanism is unknown. We aimed to study the mechanism of sepsis-associated ACLF using a novel mouse model. APPROACH AND RESULTS: Sepsis-associated ACLF was induced by cecal ligation and puncture procedure (CLP) in mice treated with thioacetamide (TAA). The combination of TAA and CLP resulted in a significant decrease in liver synthetic function and high mortality. These changes were associated with reduced metabolic gene expression and increased CCAAT enhancer binding protein beta (C/EBPß) transcriptional activity. We found that C/EBPß binding to its target gene promoters was increased. In humans, C/EBPß chromatin binding was similarly increased in the ACLF group compared with control cirrhosis. Hepatocyte-specific Cebpb knockout mice had reduced mortality and increased gene expression of hepatocyte differentiation markers in TAA/CLP mice, suggesting that C/EBPß promotes liver failure in these mice. C/EBPß activation was associated with endothelial dysfunction, characterized by reduced Angiopoietin-1/Angiopoietin-2 ratio and increased endothelial production of HGF. Angiopoietin-1 supplementation or Hgf knockdown reduced hepatocyte C/EBPß accumulation, restored liver function, and reduced mortality, suggesting that endothelial dysfunction induced by sepsis drives ACLF through HGF-C/EBPß pathway. CONCLUSIONS: The transcription factor C/EBPß is activated in both mouse and human ACLF and is a potential therapeutic target to prevent liver failure in patients with sepsis and cirrhosis.

Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression.

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

Differential Associations of Circulating MicroRNAs With Pathogenic Factors in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous disease driven by genetic and environmental factors. MicroRNAs (miRNAs) serve as pleiotropic post-transcriptional regulators of cellular pathways. Although several miRNAs have been associated with NAFLD and fibrosis, there are limited studies in humans examining their differential association with pathogenic factors or histological features of NAFLD. We examined the differential relationships of five of the best-described circulating microRNAs (miR-34a, miR-122, miR-191, miR-192, and miR-200a) with histological features and pathogenic factors of NAFLD. A cross-sectional study was conducted to examine the relationship between relative levels of circulating microRNAs standardized by z-scores and histological features of NAFLD, common NAFLD genetic polymorphisms, and insulin resistance measured by the enhanced lipoprotein insulin resistance index in 132 subjects with biopsy-proven NAFLD. We found that miR-34a, miR-122, miR-192, miR-200a, but not miR-191, strongly correlate with fibrosis in NAFLD by increases of 0.20 to 0.40 SD (P < 0.005) with each stage of fibrosis. In multivariate analysis, miR-34a, miR-122, and miR-192 levels are independently associated with hepatic steatosis and fibrosis, but not lobular inflammation or ballooning degeneration, whereas miR-200a is only associated with fibrosis. Among the four miRNAs, miR-34a, miR-122, and miR-192 are associated with pathogenic factors of NAFLD, including insulin resistance measured by eLP-IR, patatin-like phospholipase domain containing 3 I148M, and transmembrane 6 superfamily 2 (TM6SF2) E167K polymorphisms. In contrast, miR-200a is only associated with the TM6SF2 E167K variant. Finally, miR-34a has the strongest predictive value for various stages of fibrosis, with C-statistic approximates-combined predictive score for miRNAs. Conclusion: miR-34a, miR-122, miR-192, and miR-200a demonstrate strong associations with NAFLD severity by histology, but differential associations with pathogenic factors.

Hoxa1 targets signaling pathways during neural differentiation of ES cells and mouse embryogenesis.

Hoxa1 has important functional roles in neural crest specification, hindbrain patterning and heart and ear development, yet the enhancers and genes that are targeted by Hoxa1 are largely unknown. In this study, we performed a comprehensive analysis of Hoxa1 target genes using genome-wide Hoxa1 binding data in mouse ES cells differentiated with retinoic acid (RA) into neural fates in combination with differential gene expression analysis in Hoxa1 gain- and loss-of-function mouse and zebrafish embryos. Our analyses reveal that Hoxa1-bound regions show epigenetic marks of enhancers, occupancy of Hox cofactors and differential expression of nearby genes, suggesting that these regions are enriched for enhancers. In support of this, 80 of them mapped to regions with known reporter activity in transgenic mouse embryos based on the Vista enhancer database. Two additional enhancers in Dok5 and Wls1 were shown to mediate neural expression in developing mouse and zebrafish. Overall, our analysis of the putative target genes indicate that Hoxa1 has input to components of major signaling pathways, including Wnt, TGF-ß, Hedgehog and Hippo, and frequently does so by targeting multiple components of a pathway such as secreted inhibitors, ligands, receptors and down-stream components. We also identified genes implicated in heart and ear development, neural crest migration and neuronal patterning and differentiation, which may underlie major Hoxa1 mutant phenotypes. Finally, we found evidence for a high degree of evolutionary conservation of many binding regions and downstream targets of Hoxa1 between mouse and zebrafish. Our genome-wide analyses in ES cells suggests that we have enriched for in vivo relevant target genes and pathways associated with functional roles of Hoxa1 in mouse development.

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.

Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3' enhancer of Hoxa1 This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.

Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.

The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.

Illicit Use of Prescription Opiates among Graduate Students.

Through this study the authors assessed the prevalence rate, reasons for use, and poly-substance use of prescription opiates among graduate students. The authors employed a cross-sectional survey research design using an online, self-administered questionnaire to assess the prevalence rates of prescription opiate use among graduate students (N = 1,033), reasons for use, and their likelihood for poly-substance use. The survey was e-mailed to 5,000 graduate students. Graduate students (19.7%) reported illicit use of prescription opiates in their lifetime and 6.6% reported past-year illicit use. Those who indicated illicitly using prescription opiates did so for self-medication reasons; a few respondents indicated recreational use. Students using prescription opiates were 75% less likely to use marijuana; 79% less likely to use cocaine; and 75% less likely to use ecstasy. Graduate students are illicitly using prescription opiates, but primarily for self-medication, and, while doing so, are less likely to use other substances.

Hox complex analysis through BAC recombineering.

BAC transgenesis in mice has proved to be useful in exploring the regulatory mechanisms and functions of the Hox complexes. The large constructs used may include most of the relevant components of the cis-regulatory landscape. Manipulations can be accomplished without compromising the integrity of the endogenous complex which reduces the likelihood of producing confounding phenotypic abnormalities. The development of recombineering tools has been critical in providing the means necessary to make many types of precise and varied manipulations of these large constructs. Here, we will discuss the methodologies necessary to manipulate Hox complex BACs, generation of transgenic animals bearing these constructs and the utilization of these resources to address fundamental aspects of Hox biology.

Invited commentary: Fostering resilience among Native American youth through therapeutic intervention.

This article offers a comprehensive overview and understanding of the needs of Native American Youth for researchers, educators, and practitioners based on current research and practice. Strengths and protective factors are discussed in terms of Native strengths in context, the strengths and resilience of Native ways, Indigenous ways of knowing, the relationship between cultural identity and the tribal nation, the importance of family, the roles of the wisdom keepers, spiritual ways, and communication styles. Contextual influences are explored in terms of the relationship between history and healing from intergenerational grief and trauma, the influence of acculturation, as well as current social, economic, and political issues that affect Native youth. Implications for research and therapeutic intervention are explored in terms of healing from historical trauma and oppression. The authors offer an overview of common presenting issues and recommendations, practical tribally-specific interventions, and reflections on what it means to work from a social justice and client/community advocacy perspective with a focus on providing effective therapeutic, culturally-based interventions with Native children and adolescents that promote resilience and foster positive development with this population.

A randomized, double-blind trial of lisinopril and losartan for the treatment of cardiomyopathy in duchenne muscular dystrophy.

OBJECTIVES: This study sought to compare the effectiveness and safety of an angiotensin converting enzyme inhibitor (ACE-I) (lisinopril) vs. an angiotensin receptor blocker (ARB) (losartan) for the treatment of cardiomyopathy (CM) in boys with Duchenne muscular dystrophy (DMD). BACKGROUND: Development of CM is universal in boys with DMD. ACE-I and ARB have both been suggested as effective treatment options. ARBs have been associated with skeletal muscle regeneration in a mouse model of DMD. The question of which, if either, is more effective for CM treatment in DMD remains. The purpose of this multicenter double-blind prospective study was to compare efficacy and safety of lisinopril versus losartan in the treatment of newly diagnosed CM in boys with DMD. METHODS: Echocardiographic technician inter- and intraobserver variability were tested on 2 separate days on 2 different boys with DMD CM. Results were compared with paired t-testing. Twenty-two boys with newly diagnosed DMD CM (echocardiographic ejection fraction (EF) 10% EF drop. Three boys in the aCE-I group had 3 visits, due to study funding termination. Two were withdrawn because of low EF. All their data are included in the analysis for as long as they remained in the study. Mean EF's were similar at baseline (47.5%- ACE-I, 48.4%- ARB). After 1 year each group significantly improved to 54.6% and 55.2% respectively (p=.02). There was no difference between the 2 treatment groups at 1 year. CONCLUSIONS: Inter-observer and intra-observer reliability studies showed no differences between echocardiographers on serial examinations. EF improved equally in the two groups. There is no therapeutic difference in EF improvement between lisinopril and losartan over the one-year duration for treatment of boys with DMD-related CM. TRIAL REGISTRATION: ClinicalTrials.gov NCT01982695.

BAC modification through serial or simultaneous use of CRE/Lox technology.

Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation.

cDNA targets improve whole blood gene expression profiling and enhance detection of pharmocodynamic biomarkers: a quantitative platform analysis.

BACKGROUND: Genome-wide gene expression profiling of whole blood is an attractive method for discovery of biomarkers due to its non-invasiveness, simple clinical site processing and rich biological content. Except for a few successes, this technology has not yet matured enough to reach its full potential of identifying biomarkers useful for clinical prognostic and diagnostic applications or in monitoring patient response to therapeutic intervention. A variety of technical problems have hampered efforts to utilize this technology for identification of biomarkers. One significant hurdle has been the high and variable concentrations of globin transcripts in whole blood total RNA potentially resulting in non-specific probe binding and high background. In this study, we investigated and quantified the power of three whole blood profiling approaches to detect meaningful biological expression patterns. METHODS: To compare and quantify the impact of different mitigation technologies, we used a globin transcript spike-in strategy to synthetically generate a globin-induced signature and then mitigate it with the three different technologies. Biological differences, in globin transcript spiked samples, were modeled by supplementing with either 1% of liver or 1% brain total RNA. In order to demonstrate the biological utility of a robust globin artifact mitigation strategy in biomarker discovery, we treated whole blood ex vivo with suberoylanilide hydroxamic acid (SAHA) and compared the overlap between the obtained signatures and signatures of a known biomarker derived from SAHA-treated cell lines and PBMCs of SAHA-treated patients. RESULTS: We found cDNA hybridization targets detect at least 20 times more specific differentially expressed signatures (2597) between 1% liver and 1% brain in globin-supplemented samples than the PNA (117) or no treatment (97) method at FDR = 10% and p-value < 3x10-3. In addition, we found that the ex vivo derived gene expression profile was highly concordant with that of the previously identified SAHA pharmacodynamic biomarkers. CONCLUSIONS: We conclude that an amplification method for gene expression profiling employing cDNA targets effectively mitigates the negative impact on data of abundant globin transcripts and greatly improves the ability to identify relevant gene expression based pharmacodynamic biomarkers from whole blood.

Multi-tissue coexpression networks reveal unexpected subnetworks associated with disease.

BACKGROUND: Obesity is a particularly complex disease that at least partially involves genetic and environmental perturbations to gene-networks connecting the hypothalamus and several metabolic tissues, resulting in an energy imbalance at the systems level. RESULTS: To provide an inter-tissue view of obesity with respect to molecular states that are associated with physiological states, we developed a framework for constructing tissue-to-tissue coexpression networks between genes in the hypothalamus, liver or adipose tissue. These networks have a scale-free architecture and are strikingly independent of gene-gene coexpression networks that are constructed from more standard analyses of single tissues. This is the first systematic effort to study inter-tissue relationships and highlights genes in the hypothalamus that act as information relays in the control of peripheral tissues in obese mice. The subnetworks identified as specific to tissue-to-tissue interactions are enriched in genes that have obesity-relevant biological functions such as circadian rhythm, energy balance, stress response, or immune response. CONCLUSIONS: Tissue-to-tissue networks enable the identification of disease-specific genes that respond to changes induced by different tissues and they also provide unique details regarding candidate genes for obesity that are identified in genome-wide association studies. Identifying such genes from single tissue analyses would be difficult or impossible.

Sall3 is required for the terminal maturation of olfactory glomerular interneurons.

Sall3 is a zinc finger containing putative transcription factor and a member of the Sall gene family. Members of the Sall gene family are highly expressed during development. Sall3-deficient mice die in the perinatal period because of dehydration and display alterations in palate formation and cranial nerve formation (Parrish et al. [2004] Mol Cell Biol 24:7102-7112). We examined the role of Sall3 in the development of the olfactory system. We determined that Sall3 is expressed by cells in the olfactory epithelium and olfactory bulb. Sall3 deficiency specifically alters formation of the glomerular layer. The glomerular layer was hypocellular, because of a decrease in the number of interneurons. The lateral ganglionic eminence and rostral migratory stream developed normally in Sall3-deficient animals, which suggests that Sall3 is not required for the initial specification of olfactory bulb interneurons. Fewer GAD65/67-, Pax6-, calretinin-, and calbindin-positive cells were detected in the glomerular layer, accompanied by an increase in cells positive for these markers in the granule cell layer. In addition, a complete absence of tyrosine hydroxylase expression was observed in the olfactory bulb in the absence of Sall3. However, expression of Nurr1, a marker of dopaminergic precursors, was maintained, indicating that dopaminergic precursors were present. Our data suggest that Sall3 is required for the terminal maturation of neurons destined for the glomerular layer.

A rare lethal combination of premature closure of the foramen ovale and d-transposition of the great arteries with intact ventricular septum.

We report an infant who presented with cyanosis after birth and was unresponsive to all resuscitative efforts. Emergent echocardiogram showed d-transposition of the great arteries (d-TGA) with an intact ventricular septum. Attempts to perform a bedside balloon atrial septostomy failed due to inability to traverse the atrial septum. Autopsy revealed d-TGA with intact atrial and ventricular septa and patent ductus arteriosus. Though no survivors have been reported for this rare lethal combination of lesions thus far in the literature, we speculate that recent advances in fetal treatment may soon be applicable to infants with this condition.

Endoplasmic reticulum-associated degradation is required for cold adaptation and regulation of sterol biosynthesis in the yeast Saccharomyces cerevisiae.

Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Delta cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.

Regulation of gene expression by lithium and depletion of inositol in slices of adult rat cortex.

Lithium inhibits inositol monophosphatase at therapeutically effective concentrations, and it has been hypothesized that depletion of brain inositol levels is an important chemical alteration for lithium's therapeutic efficacy in bipolar disorder. We have employed adult rat cortical slices as a model to investigate the gene regulatory consequences of inositol depletion effected by lithium using cytidine diphosphoryl-diacylglycerol as a functionally relevant biochemical marker to define treatment conditions. Genes coding for the neuropeptide hormone pituitary adenylate cyclase activating polypeptide (PACAP) and the enzyme that processes PACAP's precursor to the mature form, peptidylglycine alpha-amidating monooxygenase, were upregulated by inositol depletion. Previous work has shown that PACAP can increase tyrosine hydroxylase (TH) activity and dopamine release, and we found that the gene for GTP cyclohydrolase, which effectively regulates TH through synthesis of tetrahydrobiopterin, was also upregulated by inositol depletion. We propose that modulation of brain PACAP signaling might represent a new opportunity in the treatment of bipolar disorder.