Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

HTR7 Mediates Serotonergic Acute and Chronic Itch.

Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma.

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (µ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by µ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.

Dopaminergic hypofunctions and prepulse inhibition deficits in mice lacking midkine.

Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in various biological phenomena such as cell migration, neurogenesis, and tissue repair. We previously demonstrated that midkine-deficient (Mdk(-/-)) mice exhibited a delayed hippocampal development with impaired working memory and increased anxiety only at the age of 4 weeks. To assess whether midkine gene could play important roles in development and maintenance of central nervous system, we investigated biochemical and behavioral parameters in dopamine and glutamate neurotransmission of Mdk(-/-) mice. The Mdk(-/-) mice exhibited a hypodopaminergic state (i.e., decreased levels of dopamine and its receptors in the striatum) with no alterations of glutamatergic system (i.e., normal level of glutamate, glutamine, glycine, d-serine, l-serine, and NMDA receptors in the frontal cortex and hippocampus). We also found prepulse inhibition deficits reversed by clozapine and haloperidol in the Mdk(-/-) mice. Our results suggested that midkine deficiency may be related to neurochemical and behavioral dysfunctions in dopaminergic system.

Strain specific synaptic modifications on ventral tegmental area dopamine neurons after ethanol exposure.

BACKGROUND: Genetic factors and previous alcohol experience influence alcohol consumption in both humans and rodents. Specifically, a prior experience with ethanol increases ethanol intake in both ethanol-preferring C57BL/6 (C57) and ethanol non-preferring DBA/2 (DBA) mice. Whereas the ventral tegmental area (VTA) importantly regulates dopamine levels and ethanol intake, it is unknown whether ethanol experience differentially alters synaptic properties of VTA dopamine neurons in ethanol-preferring and non-preferring mice. METHODS: The properties of excitatory and inhibitory inputs and the ability to elicit long-term potentiation (LTP) were assessed with whole-cell patch-clamp recordings in VTA dopamine neurons from C57 and DBA mice 24 hours after a single ethanol (2 g/kg, IP) or equivalent saline injection. RESULTS: Ethanol exposure increased gamma-aminobutyric acid (GABA) release onto VTA dopamine neurons in DBA mice, as previously observed in C57 mice. However, a single ethanol exposure reduced alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) function and LTP in VTA dopamine neurons from DBA but not C57 mice. CONCLUSIONS: A single ethanol exposure selectively reduced glutamate receptor function in VTA dopamine neurons from the ethanol non-preferring DBA strain but enhanced GABA signaling in both C57 and DBA strains. These results support the notion that VTA dopamine neurons are a central target of ethanol-induced neural plasticity, which could contribute to ethanol consumption. Furthermore, these findings highlight the possible need for specialized therapeutic interventions for alcoholism based on individual intrinsic differences.

Regulation and biochemistry of mouse molybdo-flavoenzymes. The DBA/2 mouse is selectively deficient in the expression of aldehyde oxidase homologues 1 and 2 and represents a unique source for the purification and characterization of aldehyde oxidase.

Mouse molybdo-flavoenzymes consist of xanthine oxidoreductase, aldehyde oxidase (AOX1), and two recently identified proteins, AOH1 and AOH2 (aldehyde oxidase homologues 1 and 2). Here we demonstrate that CD-1, C57BL/6, 129/Sv, and other mouse strains synthesize high levels of AOH1 in the liver and AOH2 in the skin. By contrast, the DBA/2 and CBA strains are unique, having a selective deficit in the expression of the AOH1 and AOH2 genes. DBA/2 animals synthesize trace amounts of a catalytically active AOH1 protein. However, relative to CD-1 animals, an over 2 log reduction in the steady-state levels of liver AOH1 mRNA, protein, and enzymatic activity is observed in basal conditions and following administration of testosterone. The DBA/2 mouse represents a unique opportunity to purify AOX1 and compare its enzymatic characteristics to those of the AOH1 protein. The spectroscopy and biochemistry of AOX1 are very similar to those of AOH1 except for a differential sensitivity to the non-competitive inhibitory effect of norharmane. AOX1 and AOH1 oxidize an overlapping set of aldehydes and heterocycles. For most compounds, the substrate efficiency (V(max)/K(m)) of AOX1 is superior to that of AOH1. Alkylic alcohols and acetaldehyde, the toxic metabolite of ethanol, are poor substrates of both enzymes. Consistent with this, the levels of acetaldehyde in the livers of ethanol administered CD-1 and DBA/2 mice are similar, indicating that neither enzyme is involved in the in vivo biotransformation of acetaldehyde.

Alpha7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons.

C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the alpha7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in alpha7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [(125)I]alpha-bungarotoxin. However, the distribution of the [(125)I]alpha-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [(125)I]alpha-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the alpha7 receptor gene. The distribution of [(125)I]alpha-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the alpha7 gene. These data suggest that the alpha7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.

Opposite genotype-dependent mesocorticolimbic dopamine response to stress.

Identification of relevant phenotypes related to neural functioning has yet to receive the needed attention, although behavioral phenotyping, through comparative studies in inbred strains of mice, has produced some major findings (Cabib et al., 2000; Crabbe, 1999; Gerlai, 1996; Lathe, 1996). Central responses to stress play a major psychopathogenic role in the presence of a genetic liability (Fowels, 1992), and mesocortical and mesoacumbens dopamine metabolism and release are the most relevant among these responses (Abercrombie et al., 1989; Cabib and Puglisi-Allegra, 1994; Chrapusta et al., 1997; Di Chiara et al., 1999; Hervé et al., 1979; Imperato et al., 1991). Therefore, in the present study, we assessed strain-dependent differences in mesocortical and mesoaccumbens dopamine responses to a widely utilized stressful procedure (restraint), by comparing mice of the oldest and most studied inbred strains (Cabib et al., 2000): the C57BL/6JIco and DBA/2JIco. We found that stress produced inhibition of mesoaccumbens dopamine release accompanied by a very fast and strong activation of mesocortical dopamine metabolism in C57BL/6JIco mice, and the opposite in mice of the DBA/2JIco strain. These results suggest a genetic control over the balance between mesocortical and mesoaccumbens dopamine responses to stress, and provide a model for pre-clinical studies on molecular genetics of depression.

Basal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice.

In order to investigate the involvement of the opioid and nociceptin/orphanin FQ (N/OFQ) system in alcohol drinking behaviour, N/OFQ and the opioid peptides dynorphin B (DYNB) and Met-enkephalin-Arg(6) Phe(7) (MEAP) were examined in the alcohol-preferring C57BL/6J mice. Basal peptide levels were compared in the brain and the pituitary gland with basal levels in the alcohol-avoiding DBA/2J mice. Furthermore, the effects of chronic alcohol self-administration on peptides were studied in the C57BL/6J mice. Compared to the DBA/2J mice, C57BL/6J mice had low immunoreactive (ir) levels of DYNB and MEAP in the nucleus accumbens, the hippocampus, and the substantia nigra, low ir-DYNB levels in the striatum and low ir-MEAP levels in the frontal cortex. Higher ir-DYNB levels in the pituitary gland and in the periaqueductal gray (PAG) and higher ir-N/OFQ levels in the frontal cortex and in the hippocampus were detected in C57BL/6J mice compared to the DBA/2J mice. After 4 weeks of voluntary alcohol consumption, only minor changes in steady-state peptide levels were identified. However, 5 days after the alcohol-drinking period, lower levels of all peptides were detected in the ventral tegmental area and ir-DYNB levels were also lower in the amygdala and in the substantia nigra. Twenty-one days after cessation of alcohol self-administration, the opioid peptides in alcohol-consuming C57BL/6J mice were lower in the PAG, the N/OFQ was lower in the frontal cortex and DYNB was higher in the amygdala and substantia nigra as compared to control C57BL/6J mice. This study demonstrates strain differences between C57BL/6J mice and DBA/2J mice that could contribute to divergent drug-taking behaviour, and it also demonstrates time- and structure-specific changes in neuropeptide levels after alcohol self-administration in the C57BL/6J mice.

Stress promotes major changes in dopamine receptor densities within the mesoaccumbens and nigrostriatal systems.

This study investigated the effects of stress on brain dopamine receptor densities in two inbred strains of mice. Analysis of [3H]SCH23390 binding by quantitative autoradiography revealed that repeated restraint stress significantly increases D1-like receptor density in the nucleus accumbens of mice of the DBA/2 strain whist reducing it in the caudate-putamen of C57BL/6 mice. No significant changes in D2-like receptor quantified by [3H](-)-sulpiride binding were observed in caudate, substantia nigra and accumbens of stressed C57BL/6 mice. Instead, in DBA/2 mice, stress significantly increased D2-like receptor density in the nucleus accumbens whilst reducing it in the substantia nigra. Finally, stress significantly increased D2-like receptor density within the ventral tegmental area of C57BL/6 mice whilst significantly reducing it in mice of the DBA/2 strain. These results indicate that stress promotes major changes in mesoaccumbens and nigrostriatal dopamine receptor densities. The direction of these changes depends on receptor subtype, brain area and strain. Moreover, the opposite changes of D2-like receptor densities promoted by stress in the ventral tegmental area of the two inbred strains of mice suggest that mesoaccumbens dopamine autoreceptors density might be controlled by a major genotype x stress interaction.

Coexpression of Ah receptor and CYP1A1 in hepatocytes of C57BL/6J and DBA/2J mice.

The distribution of the hepatic aryl hydrocarbon receptor (AhR) and CYP1A1 was investigated in control and 3-methylcholanthrene (3-MC)-treated C57BL/6J (B6) and DBA/2J (D2) mice. The AhR was identified as protein bands of 95 and 104 kDa in cytosol of B6 and D2 mice, respectively, while a protein of 93 kDa was detected in nuclear extracts from both murine strains. CYP1A1 was recognized as a protein of 56 kDa in microsomes from B6 and D2 mice. Lower amounts of immunodetectable AhR and CYP1A1 were observed in D2 mice, compared to B6 mice. Immunohistochemical studies were used to colocalize the AhR and CYP1A1 in adjacent liver sections. Staining for the AhR was localized most prominently in the cytoplasm of centrilobular hepatocytes and was minimal in the nuclei. Periportal hepatocytes were also reactive for the AhR, but the staining was usually localized in the nuclei. Other hepatocytes contained moderate amounts of cytoplasmic staining, whereas nuclear staining was present at low levels. Immunodetectable CYP1A1 also predominated in centrilobular hepatocytes and was negligible in periportal hepatocytes. The staining was exclusively cytoplasmic and was not seen in the nuclei of hepatocytes. Observations of adjacent liver sections revealed that the AhR and CYP1A1 were coexpressed in individual hepatocytes. A similar regional distribution for the AhR and CYP1A1 was manifested in B6 and D2 mice, with the exception that staining was more pronounced in the former under both control and induced conditions. These results showed heterogeneity in the distribution of the AhR and CYP1A1, with comparable levels of both proteins residing in the same hepatocytes. They further demonstrated that the AhR is localized in both the cytoplasm and nuclei of hepatocytes.

CRH-R1 mRNA expression in two strains of inbred mice and its regulation after repeated restraint stress.

Using in-situ hybridization histochemistry we investigated the distribution of CRH receptor 1 (CRH-R1) mRNA in the cortex of C57BL/6 (C57) and DBA/2 (DBA) strains and its regulation after repeated restraint stress. We show that DBA mice have a higher concentration of CRH-R1 mRNA than C57 mice. Repeated restraint stress produced an increase in CRH-R1 mRNA expression of C57 mice, whereas it was uneffective in DBA mice.

Strain-dependent effects of dopamine agonists on acetylcholine release in the hippocampus: an in vivo study in mice.

The effects of selective D1 or D2 dopamine receptor agonists and the indirect dopamine agonist cocaine on hippocampal acetylcholine release in mice of the C57BL/6 and DBA/2 inbred strains were investigated using intracerebral microdialysis. The D1 SKF 38393 (10, 20, 30 mg/kg, i.p.), the D2 agonist LY 171555 (0.5, 1, 2 mg/kg, i.p.) and cocaine (5, 10, 15 mg/kg, i.p.) all increased, dose-dependently, acetylcholine release in the hippocampus of C57BL/6 mice. Both the D1 agonist and cocaine did not produce any significant effect in DBA/2 mice. In the latter strain, however, LY 171555 produced a decrease in acetylcholine release that was evident after 60 min from injection of the doses of 0.5 and 1 mg/kg, but not at the dose of 2 mg/kg. The effects observed in C57BL/6 mice as well as those produced by low doses of LY 171555 in the DBA/2 strain were consistent with previous results obtained in rats. The present results indicate major strain-dependent differences in the effects of dopamine agonists on hippocampal acetylcholine release in mice. Moreover, they suggest a complex genotype-related neural organization of dopamine-acetylcholine interactions in the mesolimbic system. Finally, the strain differences in the effects of the dopamine agonists on hippocampal acetylcholine release parallel previously reported strain differences in the effects of these substances on memory consolidation.

Sex- and strain-related differences in first-pass alcohol metabolism in mice.

Adult males and females of three strains of mice, C57BL/10J, C57BL/6J and DBA/2J, were intubated or injected intraperitoneally with 0.02 ml/g body weight of a 25% alcohol solution. Thirty minutes later, their blood alcohol levels (BAL) were measured. Another group of mice, including both sexes, representative of the three strains, was fasted for 12 h and sacrificed; their stomachs were removed, homogenized, and assayed for gastric alcohol dehydrogenase (GAD) activity. Higher BALs were found in all intubated females compared to the intubated males. The reverse was observed in the injected group, which showed the males with the highest BAL values. GAD activity was evidenced in both sexes of the three strains and it was highest in the males. Strain-related differences were evident in the intubated groups and not in the injected groups. Intubated DBA animals had the lowest BALs as well as the highest GAD values. The results provide evidence for first-pass alcohol metabolism in mice and show the effects of sex and strain on gastric oxidation of alcohol.

Immunocytochemical study of cytochrome P450 (1A1/1A2) induction in murine ocular tissues.

C57BL/6 and DBA/2 mice were injected intraperitoneally with beta-naphthoflavone in corn oil and killed 48 hr later. Control animals received an injection of corn oil. The immunoreactivity of cytochrome P450 1A1/1A2 expressed in different ocular tissues and liver was examined with goat anti-P450 antibody (primary antibody) and gold-conjugated anti-goat antibody (secondary antibody). DBA/2 mice, which are non-responsive to aryl hydrocarbon treatment, showed negligible levels of immunoreactivity toward anti-P450 1A1/1A2 antibody in all ocular tissues, whether or not the animals were treated with beta-naphthoflavone. In responsive C57BL/6 mice, however, the immunoreactivity of the uveal tissues, especially ciliary non-pigmented epithelium, was markedly increased by beta-naphthoflavone treatment. The time course of induction of P450 1A1/1A2 immunoreactivity was very similar for the liver and ciliary non-pigmented epithelium, although the maximum level of immunoreactivity of the ciliary epithelium reached in 48 hr after inducer treatment was about 25% of that of liver. The present results support our previous observations that the P4501A enzyme activities (e.g. aryl hydrocarbon hydroxylase) in the liver and eye of C57BL/6 mice are under the same genetic regulation. Further, this study is the first demonstration of P450 isoform induction in specific ocular tissues of the whole animal.

Regulation of cytochrome P-450 isozymes by methylenedioxyphenyl compounds.

Regulation of cytochrome P-450 isozymes 1a-1, 1a-2, and 2b-10 by methylenedioxyphenyl compounds was studied by measuring levels of mRNA, protein, and enzyme activity in hepatic tissue from C57BL/6 (Ah+) and DBA/2 (Ah-) mice dosed with isosafrole (ISO) or piperonyl butoxide (PBO). Increases in 1a-2 and 2b-10 protein were observed for ISO and PBO in both strains of mice, suggesting an Ah receptor-independent mechanism for induction of these isozymes; 1a-1 induction, however, was seen only in C57 mice. Piperonyl butoxide was the more potent inducing agent in both strains. In C57 mice treated with five dose levels of PBO, induction of 1a-1 mRNA, protein, and enzyme activity were seen at doses equal to or greater than 104 mg/kg, but were not detected at lower doses. With isosafrole, induction of 1a-1 mRNA was observed only at the highest dose tested (400 mg/kg); however, neither 1a-1 protein nor increased enzymatic activity was seen at this dose. Dose-response studies showed maximum inducible levels for 1a-2 and 2b-10 protein, beyond which the mRNAs continued to increase while the protein levels remained constant.

Two-dimensional electrophoretic protein patterns of reciprocal hybrids of the mouse strains DBA and C57BL.

Two-dimensional electrophoretic patterns of cytoplasmic liver proteins of the mouse were investigated in reciprocal hybrids of the two inbred mouse strains DBA and C57BL in order to establish whether strain-specific protein variants reveal a mode of inheritance compatible with the concept of genomic imprinting. Variants of this type were found to account for about 11% of approximately 200 variant protein spots scrutinized. Transmission of the maternal form of a variant protein was more frequent than transmission of the paternal form. Maternal/paternal transmission was observed only for proteins showing strain variations in their amount. The results are discussed in terms of the frequency of imprinted genes.

In vitro brain acetylcholinesterase response among three inbred strains of mice to monocrotophos.

The strain differences in the neurotoxic potential of monocrotophos (MCP) were assessed by determining the inhibition of brain acetylcholinesterase (AChE) in BALB/cAnN, DBA/2J and C57BL/6J in vitro. MCP being a competitive inhibitor for AChE, alters the Km values widely among these inbred strains. Comparatively least alterations in Km were found in BALB/cAnN and maximum in DBA/2J. Based on the Ki values DBA/2J was found to be the most sensitive strain to MCP inhibition followed by C57BL/6J and BALB/cAnN.

Age-related cholinergic drug effects on analgesia in two inbred strains of mice.

The two inbred strains of mice C57BL/6 and DBA/2 are characterized by a different behavioral reactivity to cholinergic agents during development. The present experiment revealed that the strain-dependent differences in cholinergic-mediated analgesia during development disappeared during adult life. In fact, oxotremorine administration (0.0025 and 0.005 mg/kg) exerted the same analgesic effect in both strains at 6 months of age, in contrast with the finding of the lack of any effect of the drug in C57 mice at two months of age in comparison with DBA.