Pharmacokinetics and Tolerability of a Single Dose of Apraglutide, a Novel, Long-Acting, Synthetic glucagon-like peptide-2 Analog With a Unique Pharmacologic Profile, in Individuals With Impaired Renal Function.

Renal impairment is a common complication in patients with short bowel syndrome with intestinal failure (SBS-IF). Glucagon-like peptide-2 analogs, such as apraglutide, have been developed as a treatment option for SBS-IF. This study assessed the potential for apraglutide overexposure in individuals with severely impaired renal function versus healthy volunteers with normal renal function. In this phase 1, open-label, multicenter, nonrandomized, parallel-group study, a single dose of apraglutide 5 mg was administered subcutaneously to individuals with severely impaired renal function (<30 mL/min/1.73 m2) and healthy volunteers with normal renal function (≥90 mL/min/1.73 m2). Primary pharmacokinetic endpoints were maximum observed concentration (Cmax) and exposure to apraglutide (area under the curve [AUC] from time 0 to infinity [AUCinf], and AUC from time 0 to the last quantifiable concentration [AUClast]). Each group comprised 8 individuals. Results show that patients with severe renal impairment do not have increased apraglutide exposure. Apraglutide achieved a lower Cmax and AUCinf in individuals with severe renal impairment versus those with normal renal function (Cmax = 36.9 vs 59.5 ng/L; AUCinf = 3100 vs 4470 h · ng/mL, respectively). The respective geometric mean ratios were 0.620 and 0.693 for Cmax and AUCinf, and the upper bound of their 90% confidence intervals were <2, indicating patients with severe renal impairment were not overexposed to apraglutide versus those with normal renal function. Adverse events were mild or moderate in severity. Apraglutide does not require dose reduction for any degree of renal impairment and could be used in a broader patient population of renally impaired patients without dose adjustment.

Characterization of the Pharmacokinetic and Pharmacodynamic Profile of Apraglutide, a Glucagon-Like Peptide-2 Analog, in Healthy Volunteers.

Apraglutide (FE 203799) is a glucagon-like peptide-2 (GLP-2) analog under development for the treatment of intestinal failure associated with short bowel syndrome (SBS-IF) and graft-versus-host disease (GvHD). Compared with native GLP-2, apraglutide has slower absorption, reduced clearance, and higher protein binding, enabling once-weekly dosing. This study evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) profile of apraglutide in healthy adults. Healthy volunteers were randomized to receive 6 weekly subcutaneous administrations of 1, 5, or 10 mg apraglutide or placebo. PK and citrulline (an enterocyte mass PD marker) samples were collected at multiple time points. Kinetic parameters of apraglutide and citrulline were calculated using noncompartmental analysis; repeated PD measures were analyzed with a mixed model of covariance. A population PK/PD model was developed that also included data from a previous phase 1 study in healthy volunteers. Twenty-four subjects were randomized; 23 received all study drug administrations. Mean estimated apraglutide clearance was 16.5-20.7 l/day, and mean volume of distribution was 55.4-105.0 liters. A dose-dependent increase in citrulline plasma concentration was observed, with 5-mg and 10-mg doses inducing higher citrulline levels than 1-mg doses and placebo. PK/PD analysis showed that weekly 5-mg apraglutide induced the maximal citrulline response. Increased plasma citrulline levels were sustained for 10-17 days after the final apraglutide administration. Apraglutide displays predictable dose-dependent PK and PD profiles, with a 5-mg dose showing significant PD effects. Results suggest that apraglutide has early and enduring effects on enterocyte mass and supports the continued development of weekly subcutaneous apraglutide for SBS-IF and GvHD patient populations. SIGNIFICANCE STATEMENT: Once-weekly subcutaneous apraglutide results in dose-dependent elevations of plasma citrulline (an enterocyte mass pharmacodynamic marker) with parameters suggesting that apraglutide has lasting effects on enterocyte mass and the potential to provide therapeutic benefits. This is the first report of a model relating glucagon-like peptide-2 (GLP-2) agonism and its effects in intestinal mucosa, affording not only the ability to predict pharmacologic effects of GLP-2 analogs but also the exploration of optimal dosing regimens for this drug class across populations with different body weights.

Apraglutide, a novel once-weekly glucagon-like peptide-2 analog, improves intestinal fluid and energy absorption in patients with short bowel syndrome: An open-label phase 1 and 2 metabolic balance trial.

BACKGROUND: Apraglutide is a novel long-acting glucagon-like peptide-2 (GLP-2) analog designed for once-weekly subcutaneous dosing, with the potential to increase fluid and nutrient absorption by the remnant intestine of patients who have short bowel syndrome (SBS) with intestinal insufficiency (SBS-II) or intestinal failure (SBS-IF). This trial investigated the safety and effects on intestinal absorption of apraglutide in patients with SBS-II and SBS-IF. METHODS: In this open-label, phase 1 and 2 trial, adult patients with SBS-II (n = 4) or SBS-IF (n = 4) and a fecal output of ≥1500 g/day received once-weekly subcutaneous 5 mg apraglutide for 4 weeks. Safety was the primary end point. Secondary end points included change from baseline in intestinal absorption of wet weight (indicative of fluid absorption), electrolytes, and energy (by bomb calorimetry) measured by inpatient metabolic balance studies. RESULTS: Common treatment-related adverse events were decreased gastrointestinal (GI) stoma output (n = 6), stoma complications (n = 6), GI stoma complications (n = 5), nausea (n = 5), flatulence (n = 4), abnormal GI stoma output (n = 4), polyuria (n = 3), and abdominal pain (n = 3). The only treatment-related serious adverse event (experienced in one patient) was abdominal pain. Apraglutide significantly increased wet weight and energy absorption by an adjusted mean of 741 g/day (95% CI, 194 to 1287; P = 0.015) and 1095 kJ/day (95% CI, 196 to 1994; P = 0.024), respectively. Sodium and potassium absorption significantly increased by an adjusted mean of 38 mmol/day (95% CI, 3 to 74; P = 0.039) and 18 mmol/day (95% CI, 4 to 32; P = 0.020), respectively. CONCLUSION: Once-weekly 5 mg apraglutide was well tolerated in patients with SBS-II and SBS-IF and significantly improved the absorption of fluids, electrolytes, and energy.

Apraglutide, a novel glucagon-like peptide-2 analog, improves fluid absorption in patients with short bowel syndrome intestinal failure: Findings from a placebo-controlled, randomized phase 2 trial.

BACKGROUND: Treatment with glucagon-like peptide-2 (GLP-2) analogs improve intestinal adaptation in patients with short bowel syndrome-associated intestinal failure (SBS-IF) and may reduce parenteral support requirements. Apraglutide is a novel, long-acting GLP-2 analog designed for once-weekly dosing. This trial investigated the safety and efficacy of apraglutide in patients with SBS-IF. METHODS: In this placebo-controlled, double-blind, randomized, crossover phase 2 trial, eight adults with SBS-IF were treated with once-weekly 5-mg apraglutide doses and placebo for 4 weeks, followed by once-weekly 10-mg apraglutide doses for 4 weeks, with a washout period of 6-10 weeks between treatments. Safety was the primary end point. Secondary end points included changes from baseline in urine volume output compared with placebo, collected for 48 h before and after each treatment period. RESULTS: Common treatment-related adverse events (AEs) were mild to moderate and included polyuria, decreased stoma output, stoma complications, decreased thirst, and edema. No serious AEs were considered to be related to apraglutide treatment. The safety profile was comparable for the lower and higher doses. Treatment with once-weekly 5- and 10-mg apraglutide doses significantly increased urine volume output by an adjusted mean of 714 ml/day (95% CI, 490-939; P < .05) and 795 ml/day (95% CI, 195-1394; P < .05), respectively, compared with placebo, with no significant differences between doses. CONCLUSIONS: Once-weekly apraglutide was well tolerated at both tested doses and significantly increased urine volume output, providing evidence for increased intestinal fluid absorption. A phase 3 trial is underway in adults with SBS-IF.

Psychiatric Comorbidities and Psychotropic Medication Use in Autism: A Matched Cohort Study with ADHD and General Population Comparator Groups in the United Kingdom.

Psychiatric comorbidities and use of psychotropic medications are common among patients with autism spectrum disorder (ASD). However, most previous research used data from the United States (US) and few studies have compared medication use in ASD to control groups, making contextualization of results difficult. In the United Kingdom (UK), general practitioners play a key role in the management of ASD. We conducted a retrospective, cross-sectional study over calendar year 2015, using primary care data from the UK. We identified a prevalent cohort of ASD cases (n = 10,856) and matched control groups of (a) general population (n = 21,712) and (b) attention deficit hyperactivity disorder (ADHD; n = 7,058) on age, sex and region. We described psychiatric comorbidities, psychotropic medications, and healthcare utilization in all three cohorts. Within the ASD cohort, we used multivariable logistic regression models to explore associations between patient characteristics and the outcomes of: any psychotropic medication, polypharmacy, and number of primary care visits. We used conditional logistic regression to compare the ASD and control groups. Psychiatric comorbidities were recorded for 41.5% of ASD patients; 32.3% received psychotropic medication and 9.8% received polypharmacy. Increased age and all psychiatric comorbidities (except conduct disorder) were associated with treatment use. Males were less likely to receive a treatment than females [Odds ratio (OR) 0.74 (0.66-0.83)]. ASD patients were more likely to take psychotropic medications than the general population [OR 4.91 (4.46-5.40)], but less likely compared to ADHD patients [OR 0.40 (0.37-0.44)]. Overall, rates of medication use in the UK were lower than those previously reported in the US. Autism Research 2018, 11: 1690-1700. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: We used electronic medical records from the UK, to describe the amount of psychiatric comorbidities, psychotropic medication use and healthcare resource use in ASD. Around one in three people with ASD were prescribed a psychotropic medication, which was more than the general population, but less than for those with ADHD. Increased age, psychiatric comorbidities and female gender were all independently associated with psychotropic medication use. Rates of medication use in the UK were lower than those previously reported in the US.

Psychiatric comorbidities and use of psychotropic medications in people with autism spectrum disorder in the United States.

This study investigated psychotropic medication usage in two large, cohorts of people with autism spectrum disorder (ASD) throughout the calendar year 2014. The cohorts referred to individuals with commercial (employer-sponsored) and Medicaid insurance in the United States. We aimed to understand prescribing patterns of such medications across a wide age-range and in the presence/absence of other clinical and non-clinical characteristics, including psychiatric comorbidities. We described the prevalence and length of prescriptions by age, psychiatric comorbidity and overall. We also fitted multivariable logistic regression models to describe the relationship between treatments and subject characteristics simultaneously. Eighty percent of the identified population was male, although gender did not impact the odds of receiving medication. Medication use was strongly associated with age, increasing most rapidly before adulthood; generally plateauing thereafter. All psychiatric comorbidities studied also individually increased the chances of medication use, with epilepsy and ADHD having the highest associations in both the commercial (OR > 7) and Medicaid (OR around 12) cohorts. Those in non-capitated insurance plans, in foster care and white individuals also had increased odds of prescriptions. Overall, slightly more Medicaid enrollees received any psychotropic treatment (commercial: 64%, Medicaid: 69%). Nonetheless in both cohorts, a large proportion of individuals received treatment even without a diagnosis of any other psychiatric comorbidity (commercial: 31%, Medicaid: 33%). In summary, this report sheds new light on the latest patterns of psychiatric comorbidity profile and psycho-pharmacological treatment patterns in ASD Autism Res 2017, 10: 2037-2047. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: this study identified a large number of children and adults in the US with autism spectrum disorder (autism) from employer-sponsored and government funded (Medicaid) health insurance data. Psychotropic medications were used by over two thirds of people, and four in ten people received two medications at the same time. The chances of receiving medication increased for individuals with other psychiatric conditions (e.g., ADHD), and also increased with age.

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for ß-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and ß-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome.

BACKGROUND: Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital. RESULTS: Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters. CONCLUSIONS: Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells.

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing adenylate/uridylate-rich element (ARE) located at the 3' untranslated region (3'UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using tissue microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer.

Increased expression of axogenesis-related genes and mossy fibre length in dentate granule cells from adult HuD overexpressor mice.

The neuronal RNA-binding protein HuD plays a critical role in the post-transcriptional regulation of short-lived mRNAs during the initial establishment and remodelling of neural connections. We have generated transgenic mice overexpressing this protein (HuD-Tg) in adult DGCs (dentate granule cells) and shown that their mossy fibres contain high levels of GAP-43 (growth-associated protein 43) and exhibit distinct morphological and electrophysiological properties. To investigate the basis for these changes and identify other molecular targets of HuD, DGCs from HuD-Tg and control mice were collected by LCM (laser capture microscopy) and RNAs analysed using DNA microarrays. Results show that 216 known mRNAs transcripts and 63 ESTs (expressed sequence tags) are significantly up-regulated in DGCs from these transgenic mice. Analyses of the 3'-UTRs (3'-untranslated regions) of these transcripts revealed an increased number of HuD-binding sites and the presence of several known instability-conferring sequences. Among these, the mRNA for TTR (transthyretin) shows the highest level of up-regulation, as confirmed by qRT-PCR (quantitative reverse transcription-PCR) and ISH (in situ hybridization). GO (gene ontology) analyses of up-regulated transcripts revealed a large over-representation of genes associated with neural development and axogenesis. In correlation with these gene expression changes, we found an increased length of the infrapyramidal mossy fibre bundle in HuD-Tg mice. These results support the notion that HuD stabilizes a number of developmentally regulated mRNAs in DGCs, resulting in increased axonal elongation.

Novel recognition motifs and biological functions of the RNA-binding protein HuD revealed by genome-wide identification of its targets.

HuD is a neuronal ELAV-like RNA-binding protein (RBP) involved in nervous system development, regeneration, and learning and memory. This protein stabilizes mRNAs by binding to AU-rich instability elements (AREs) in their 3' unstranslated regions (3' UTR). To isolate its in vivo targets, messenger ribonucleoprotein (mRNP) complexes containing HuD were first immunoprecipitated from brain extracts and directly bound mRNAs identified by subsequent GST-HuD pull downs and microarray assays. Using the 3' UTR sequences of the most enriched targets and the known sequence restrictions of the HuD ARE-binding site, we discovered three novel recognition motifs. Motifs 2 and 3 are U-rich whereas motif 1 is C-rich. In vitro binding assays indicated that HuD binds motif 3 with the highest affinity, followed by motifs 2 and 1, with less affinity. These motifs were found to be over-represented in brain mRNAs that are upregulated in HuD overexpressor mice, supporting the biological function of these sequences. Gene ontology analyses revealed that HuD targets are enriched in signaling pathways involved in neuronal differentiation and that many of these mRNAs encode other RBPs, translation factors and actin-binding proteins. These findings provide further insights into the post-transcriptional mechanisms by which HuD promotes neural development and synaptic plasticity.

Alterations in mossy fiber physiology and GAP-43 expression and function in transgenic mice overexpressing HuD.

HuD is a neuronal RNA-binding protein associated with the stabilization of mRNAs for GAP-43 and other neuronal proteins that are important for nervous system development and learning and memory mechanisms. To better understand the function of this protein, we generated transgenic mice expressing human HuD (HuD-Tg) in adult forebrain neurons. We have previously shown that expression of HuD in adult dentate granule cells results in an abnormal accumulation of GAP-43 mRNA via posttranscriptional mechanisms. Here we show that this mRNA accumulation leads to the ectopic expression of GAP-43 protein in mossy fibers. Electrophysiological analyses of the mossy fiber to CA3 synapse of HuD-Tg mice revealed increases in paired-pulse facilitation (PPF) at short interpulse intervals and no change in long-term potentiation (LTP). Presynaptic calcium transients at the same synapses exhibited faster time constants of decay, suggesting a decrease in the endogenous Ca(2+) buffer capacity of mossy fiber terminals of HuD-Tg mice. Under resting conditions, GAP-43 binds very tightly to calmodulin sequestering it and then releasing it upon PKC-dependent phosphorylation. Therefore, subsequent studies examined the extent of GAP-43 phosphorylation and its association to calmodulin. We found that despite the increased GAP-43 expression in HuD-Tg mice, the levels of PKC-phosphorylated GAP-43 were decreased in these animals. Furthermore, in agreement with the increased proportion of nonphosphorylated GAP-43, HuD-Tg mice showed increased binding of calmodulin to this protein. These results suggest that a significant amount of calmodulin may be trapped in an inactive state, unable to bind free calcium, and activate downstream signaling pathways. In conclusion, we propose that an unregulated expression of HuD disrupts mossy fiber physiology in adult mice in part by altering the expression and phosphorylation of GAP-43 and the amount of free calmodulin available at the synaptic terminal.

Coordinated expression of HuD and GAP-43 in hippocampal dentate granule cells during developmental and adult plasticity.

Previous work from our laboratory demonstrated that the RNA-binding protein HuD binds to and stabilizes the GAP-43 mRNA. In this study, we characterized the expression of HuD and GAP-43 mRNA in the hippocampus during two forms of neuronal plasticity. During post-natal development, maximal expression of both molecules was found at P5 and their levels steadily decreased thereafter. At P5, HuD was also present in the subventricular zone, where it co-localized with doublecortin. In the adult hippocampus, the basal levels of HuD and GAP-43 were lower than during development but were significantly increased in the dentate gyrus after seizures. The function of HuD in GAP-43 gene expression was confirmed using HuD-KO mice, in which the GAP-43 mRNA was significantly less stable than in wild type mice. Altogether, these results demonstrate that HuD plays a role in the post-transcriptional control of GAP-43 mRNA in dentate granule cells during developmental and adult plasticity.

The RNA-binding protein HuD binds acetylcholinesterase mRNA in neurons and regulates its expression after axotomy.

After axotomy, expression of acetylcholinesterase (AChE) is greatly reduced in the superior cervical ganglion (SCG); however, the molecular events involved in this response remain unknown. Here, we first examined AChE mRNA levels in the brain of transgenic mice that overexpress human HuD. Both in situ hybridization and reverse transcription-PCR demonstrated that AChE transcript levels were increased by more than twofold in the hippocampus of HuD transgenic mice. Additionally, direct interaction between the HuD transgene product and AChE mRNA was observed. Next, we examined the role of HuD in regulating AChE expression in intact and axotomized rat SCG neurons. After axotomy of the adult rat SCG neurons, AChE transcript levels decreased by 50 and 85% by the first and fourth day, respectively. In vitro mRNA decay assays indicated that the decrease in AChE mRNA levels resulted from changes in the stability of presynthesized transcripts. A combination of approaches performed using the region that directly encompasses an adenylate and uridylate (AU)-rich element within the AChE 3'-untranslated region demonstrated a decrease in RNA-protein complexes in response to axotomy of the SCG and, specifically, a decrease in HuD binding. After axotomy, HuD transcript and protein levels also decreased. Using a herpes simplex virus construct containing the human HuD sequence to infect SCG neurons in vivo, we found that AChE and GAP-43 mRNA levels were maintained in the SCG after axotomy. Together, the results of this study demonstrate that AChE expression in neurons of the rat SCG is regulated via post-transcriptional mechanisms that involve the AU-rich element and HuD.

Associative and spatial learning and memory deficits in transgenic mice overexpressing the RNA-binding protein HuD.

HuD is a neuronal specific RNA-binding protein associated with the stabilization of short-lived mRNAs during brain development, nerve regeneration and synaptic plasticity. To investigate the functional significance of this protein in the mature brain, we generated transgenic mice overexpressing HuD in forebrain neurons under the control of the alphaCaMKinII promoter. We have previously shown that one of the targets of HuD, GAP-43 mRNA, was stabilized in neurons in the hippocampus, amygdala and cortex of transgenic mice. Animals from two independent lines expressing different levels of the transgene were subjected to a battery of behavioral tests including contextual fear conditioning and the Morris water maze. Our results show that although HuD is increased after learning and memory, constitutive HuD overexpression impaired the acquisition and retention of both cued and contextual fear and the ability to remember the position of a hidden platform in the Morris water maze. No motor-sensory abnormalities were observed in HuD transgenic mice, suggesting that the poor performance of the mice in these tests reflect a true cognitive impairment. We conclude that posttranscriptional regulation of gene expression by stabilization of specific mRNAs may have to be restricted temporally and spatially for proper acquisition and storage of memories.

Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins.

This study was aimed at establishing whether oxidative stress induced by acute depletion of brain glutathione (GSH) is sufficient to generate protein carbonyls (PCOs). To this end, rat brain slices were incubated separately with the GSH depletors 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on Western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1 mM BCNU or 10 mM DEM for 2 hr decreased GSH levels by > 70%. Under these conditions the carbonylation of several proteins (40-120 kDa) increased by 2-3 fold. Isolation of carbonylated proteins showed that augmented PCOs represents a rise in the amount of oxidized protein. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation and lipid peroxidation (TBARS), indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H(2)O(2) (Fenton reaction). Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not increase PCOs or TBARS, suggesting that increased production of reactive oxygen species (ROS) rather than compromised cellular antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was not affected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and carbonyl cyanide 3-chlorophenylhydrazone, indicating that the microsomal monooxygenase system and the mitochondrial electron transport system are the major sources of ROS. Consistent with these findings, subcellular fractionation studies showed that mitochondria and synaptosomes are the major PCO-containing organelles. These results were also supported by the anatomic distribution of PCOs in brain. Our observations may be important in the context of multiple sclerosis where decreased GSH, mitochondrial dysfunction, excessive production of ROS, and increased protein carbonylation have all been reported.

In vivo post-transcriptional regulation of GAP-43 mRNA by overexpression of the RNA-binding protein HuD.

HuD is a neuronal-specific RNA-binding protein that binds to and stabilizes the mRNAs of growth-associated protein-43 (GAP-43) and other neuronal proteins. HuD expression increases during brain development, nerve regeneration, and learning and memory, suggesting that this protein is important for controlling gene expression during developmental and adult plasticity. To examine the function of HuD in vivo, we generated transgenic mice overexpressing human HuD under the control of the calcium-calmodulin-dependent protein kinase IIalpha promoter. The transgene was expressed at high levels throughout the forebrain, including the hippocampal formation, amygdala and cerebral cortex. Using quantitative in situ hybridization, we found that HuD overexpression led to selective increases in GAP-43 mRNA in hippocampal dentate granule cells and neurons in the lateral amygdala and layer V of the neorcortex. In contrast, GAP-43 pre-mRNA levels were unchanged or decreased in the same neuronal populations. Comparison of the levels of mature GAP-43 mRNA and pre-mRNA in the same neurons of transgenic mice suggested that HuD increased the stability of the transcript. Confirming this, mRNA decay assays revealed that the GAP-43 mRNA was more stable in brain extracts from HuD transgenic mice than non-transgenic littermates. In conclusion, our results demonstrate that HuD overexpression is sufficient to increase GAP-43 mRNA stability in vivo.

Dendritic localization of the RNA-binding protein HuD in hippocampal neurons: association with polysomes and upregulation during contextual learning.

The RNA-binding protein HuD binds to and stabilizes a number of neuronal-specific mRNAs. Recent work from our laboratory indicated that HuD expression is increased in neurons during peripheral nerve regeneration. To gain further insight into the function of this protein in CNS neurons we examined the levels of expression and localization of HuD in hippocampal neurons under normal conditions and in animals subjected to a learning paradigm, contextual fear conditioning (CFC). In the adult hippocampal formation, HuD immunoreactivity was highest in CA3 pyramidal neurons and interneurons in the hilus, moderate in the CA1 region and not detectable in dentate granule cells. Using confocal microscopy we found that HuD immunoreactivity was associated with large cytoplasmic granules in the neuronal cell body and smaller granules in dendrites. Both types of granules were also stained with the ribosomal marker Y10B, suggesting that they also contain ribosomes. Consistent with this idea, subcellular fractionation and immunoprecipitation analyses indicated that HuD is present in both the polysomal (P130) and cytosolic (S130) fraction. In addition to the basal pattern of HuD expression, we examined changes in the levels of this protein 24 h after rats were subjected to a single trial CFC paradigm. HuD protein expression was found to increase in the hilus and CA3 regions of the hippocampus but not in CA1. Our findings suggest that HuD plays a role in synaptic plasticity mechanisms stabilizing mRNAs associated with ribosomes both in the soma and dendrites of hippocampal neurons.

Role of HuD and other RNA-binding proteins in neural development and plasticity.

Transcription factors have traditionally been viewed as the main determinants of gene expression. Yet, in recent years it has become apparent that RNA-binding proteins also play a critical role in determining the levels of expression of a large number of genes. Once mRNAs are transcribed, RNA-binding proteins can control all subsequent steps in their function, from alternative splicing and translation to mRNA transport and stability. In the nervous system, a large number of genes are regulated post-transcriptionally via the interaction of their mRNAs with specific RNA-binding proteins. This type of regulation is particularly important in the control of the temporal and spatial pattern of gene expression during neural development. This review will discuss the function of the embryonic lethal abnormal vision (ELAV)/Hu family of nervous system-specific RNA-binding proteins, with a special emphasis on HuD, a member of this family that controls GAP-43 mRNA stability and expression. In addition, we will present recent findings on other neural RNA-binding proteins: the ribonucleoprotein K homology (KH)-domain proteins, Fragile X mental retardation protein (FMRP), quakinguiable protein (QKI), and Nova-1. Together with the ELAV/Hu family, these proteins are essential for proper neural development and in some cases for plasticity in the mature brain. The biological significance of these proteins is evident not only by their evolutionary conservation but also by the magnitude of problems arising from autoimmune reactions against them or from mutations affecting their expression or function.