The diagnostic role of glycosaminoglycans in pleural effusions: A pilot study

Background

While modulation of the serotonin transporter (5HTT) has shown to be a risk factor for pulmonary arterial hypertension for almost 40 years, there is a lack of in vivo data about the broad molecular effects of pulmonary inhibition of 5HTT. Previous studies have suggested effects on inflammation, proliferation, and vasoconstriction. The goal of this study was to determine which of these were supported by alterations in gene expression in serotonin transporter knockout mice.

Methods

Eight week old normoxic mice with a 5-HTT knock-out (5HTT-/-) and their heterozygote(5HTT+/-) or wild-type(5HTT+/+) littermates had right ventricular systolic pressure(RVSP) assessed, lungs collected for RNA, pooled, and used in duplicate in Affymetrix array analysis. Representative genes were confirmed by quantitative RT-PCR and western blot.

Results

RVSP was normal in all groups. Only 124 genes were reliably changed between 5HTT-/- and 5HTT+/+ mice. More than half of these were either involved in inflammatory response or muscle function and organization; in addition, some matrix, heme oxygenase, developmental, and energy metabolism genes showed altered expression. Quantitative RT-PCR for examples from each major group confirmed changes seen by array, with an intermediate level in 5HTT +/- mice.

Conclusion

These results for the first time show the in vivo effects of 5HTT knockout in lungs, and show that many of the downstream mechanisms suggested by cell culture and ex vivo experiments are also operational in vivo. This suggests that the effect of 5HTT on pulmonary vascular function arises from its impact on several systems, including vasoreactivity, proliferation, and immune function.     

The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos

Pax3 and Pax7 paralogous genes have functionally diverged in vertebrate evolution, creating opportunity for a new distribution of roles between the two genes and the evolution of novel functions. Here we focus on the regulation and function of Pax7 in the brain and neural crest of amphibian embryos, which display a different pax7 expression pattern, compared to the other vertebrates already described. Pax7 expression is restricted to the midbrain, hindbrain and anterior spinal cord, and Pax7 activity is important for maintaining the fates of these regions, by restricting otx2 expression anteriorly. In contrast, pax3 displays broader expression along the entire neuraxis and Pax3 function is important for posterior brain patterning without acting on otx2 expression. Moreover, while both genes are essential for neural crest patterning, we show that they do so using two distinct mechanisms: Pax3 acts within the ectoderm which will be induced into neural crest, while Pax7 is essential for the inducing activity of the paraxial mesoderm towards the prospective neural crest.     

Characterization of Behavioral,Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is caused by an expansion of the trinucleotide poly (CAG) tract located in exon 1 of the huntingtin (Htt) gene leading to progressive neurodegeneration in selected brain regions, and associated functional impairments in motor, cognitive, and psychiatric domains. Since the discovery of the gene mutation that causes the disease, mouse models have been developed by different strategies. Recently, a new model, the zQ175 knock-in (KI) line, was developed in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. The behavioral phenotype was characterized across the independent laboratories and important features reminiscent of human HD are observed in zQ175 mice. In the current study, we characterized the zQ175 model housed in an academic laboratory under reversed dark-light cycle, including motor function, in vivo longitudinal structural MRI imaging for brain volume, MRS for striatal metabolites, neuropathology, as well as a panel of key disease marker proteins in the striatum at different ages. Our results suggest that homozygous zQ175 mice exhibited significant brain atrophy before the motor deficits and brain metabolite changes. Altered striatal medium spiny neuronal marker, postsynaptic marker protein and complement component C1qC also characterized zQ175 mice. Our results confirmed that the zQ175 KI model is valuable in understanding of HD-like pathophysiology and evaluation of potential therapeutics. Our data also provide suggestions to select appropriate outcome measurements in preclinical studies using the zQ175 mice.     

Antidepressant Treatment Outcome Depends on the Quality of the Living Environment: A Pre-Clinical Investigation in Mice

Antidepressants represent the standard treatment for major depression. However, their efficacy is variable and incomplete. A growing number of studies suggest that the environment plays a major role in determining the efficacy of these drugs, specifically of selective serotonin reuptake inhibitors (SSRI). A recent hypothesis posits that the increase in serotonin levels induced by SSRI may not affect mood per se, but enhances neural plasticity and, consequently, renders the individual more susceptible to the influence of the environment. Thus, SSRI administration in a favorable environment would lead to a reduction of symptoms, while in a stressful environment might lead to a worse prognosis. To test this hypothesis, we treated C57BL/6 adult male mice with chronic fluoxetine while exposing them to either (i) an enriched environment, after exposure to a chronic stress period aimed at inducing a depression-like phenotype, or (ii) a stressful environment. Anhedonia, brain BDNF and circulating corticosterone levels, considered endophenotypes of depression, were investigated. Mice treated with fluoxetine in an enriched condition improved their depression-like phenotype compared to controls, displaying higher saccharin preference, higher brain BDNF levels and reduced corticosterone levels. By contrast, when chronic fluoxetine administration occurred in a stressful condition, mice showed a more distinct worsening of the depression-like profile, displaying a faster decrease of saccharin preference, lower brain BDNF levels and increased corticosterone levels. Our findings suggest that the effect of SSRI on depression-like phenotypes in mice is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.     

Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.     

Vesicoureteral reflux and other urinary tract malformations in mice compound heterozygous for Pax2 and Emx2

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. This disease group includes a spectrum of urinary tract defects including vesicoureteral reflux, duplex kidneys and other developmental defects that can be found alone or in combination. To identify new regulators of CAKUT, we tested the genetic cooperativity between several key regulators of urogenital system development in mice. We found a high incidence of urinary tract anomalies in Pax2;Emx2 compound heterozygous mice that are not found in single heterozygous mice. Pax2^+/−;Emx2^+/− mice harbor duplex systems associated with urinary tract obstruction, bifid ureter and a high penetrance of vesicoureteral reflux. Remarkably, most compound heterozygous mice refluxed at low intravesical pressure. Early analysis of Pax2^+/−;Emx2^+/− embryos point to ureter budding defects as the primary cause of urinary tract anomalies. We additionally establish Pax2 as a direct regulator of Emx2 expression in the Wolffian duct. Together, these results identify a haploinsufficient genetic combination resulting in CAKUT-like phenotype, including a high sensitivity to vesicoureteral reflux. As both genes are located on human chromosome 10q, which is lost in a proportion of VUR patients, these findings may help understand VUR and CAKUT in humans.     

Shh and Pax6 have unconventional expression patterns in embryonic morphogenesis in Sepia officinalis (Cephalopoda)

Cephalopods show a very complex nervous system, particularly derived when compared to other molluscs. In vertebrates, the setting up of the nervous system depends on genes such as Shh and Pax6. In this paper we assess Shh and Pax6 expression patterns during Sepia officinalis development by whole-mount in situ hybridization. In vertebrates, Shh has been shown to indirectly inhibit Pax6. This seems to be the case in cephalopods as the expression patterns of these genes do not overlap during S. officinalis development. Pax6 is expressed in the optic region and brain and Shh in gut structures, as already seen in vertebrates and Drosophila. Thus, both genes show expression in analogous structures in vertebrates. Surprisingly, they also exhibit unconventional expressions such as in gills for Pax6 and ganglia borders for Shh. They are also expressed in many cephalopods’ derived characters among molluscs as in arm suckers for Pax6 and beak producing tissues, nuchal organ and neural cord of the arms for Shh. This new data supports the fact that molecular control patterns have evolved with the appearance of morphological novelties in cephalopods as shown in this new model, S. officinalis.     

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.