Kim Kultima

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

BackgroundThe contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.ResultsHere, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4–1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds.ConclusionThe pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.

Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.

Proteomic Evaluation of Neonatal Exposure to 2,2′,4,4′,5-Pentabromodiphenyl Ether

Exposure to the brominated flame retardant 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–ToF–MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., α-enolase; γ-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, β subunit (Atp5b); and α-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.

Sex-dependent gene expression in early brain development of chicken embryos

BackgroundDifferentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. Genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms.ResultsUsing cDNA microarrays and real-time PCR we found gene expression differences between the male and female embryonic brain (or whole head) that may be independent of morphological differentiation of the gonads. Genes located on the sex chromosomes (ZZ in males and ZW in females) were common among the differentially expressed genes, several of which (WPKCI-8, HINT, MHM non-coding RNA) have previously been implicated in avian sex determination. A majority of the identified genes were more highly expressed in males. Three of these genes (CDK7, CCNH and BTF2-P44) encode subunits of the transcription factor IIH complex, indicating a role for this complex in neuronal differentiation.ConclusionIn conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds.

Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects.

The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.

Striatal Proteomic Analysis Suggests that First L-Dopa Dose Equates to Chronic Exposure

L-3,4-dihydroxypheylalanine (L-dopa)-induced dyskinesia represent a debilitating complication of therapy for Parkinsons disease (PD) that result from a progressive sensitization through repeated L-dopa exposures. The MPTP macaque model was used to study the proteome in dopamine-depleted striatum with and without subsequent acute and chronic L-dopa treatment using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The present data suggest that the dopamine-depleted striatum is so sensitive to de novo L-dopa treatment that the first ever administration alone would be able (i) to induce rapid post-translational modification-based proteomic changes that are specific to this first exposure and (ii), possibly, lead to irreversible protein level changes that would be not further modified by chronic L-dopa treatment. The apparent equivalence between first and chronic L-dopa administration suggests that priming would be the direct consequence of dopamine loss, the first L-dopa administrations only exacerbating the sensitization process but not inducing it.

Quantification of the Brain Proteome in Alzheimer’s Disease Using Multiplexed Mass Spectrometry

We have compared the brain proteome in the temporal neocortex between Alzheimers disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

Striatal Alterations of Secretogranin-1, Somatostatin, Prodynorphin, and Cholecystokinin Peptides in an Experimental Mouse Model of Parkinson Disease

The principal causative pathology of Parkinson disease is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta projecting to the striatum in the brain. The information regarding the expression of neuropeptides in parkinsonism is very limited. Here we have elucidated striatal neuropeptide mechanisms in experimental parkinsonism using the unilateral 6-hydroxydopamine model to degenerate dopamine neurons. A thoroughly controlled sample preparation technique together with a peptidomics approach and targeted neuropeptide sequence collections enabled sensitive detection, identification, and relative quantitation of a great number of endogenous neuropeptides. Previously not recognized alterations in neuropeptide levels were identified in the unilateral lesioned mice with or without subchronic 3,4-dihydroxy-l-phenylalanine administration, the conventional treatment of Parkinson disease. Several of these peptides originated from the same precursor such as secretogranin-1, somatostatin, prodynorphin, and cholecystokinin. Disease-related biotransformation of precursors into individual peptides was observed in the experimental model of Parkinson disease. Several previously unreported potentially biologically active peptides were also identified from the striatal samples. This study provides further evidence that neuropeptides take part in mediating the central nervous system failure associated with Parkinson disease.

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

Neurotoxin-induced neuropeptide perturbations in striatum of neonatal rats.

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9-10) with BMAA (40-460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain.