Intermittent Hypoxic Conditioning Rescues Cognition and Mitochondrial Bioenergetic Profile in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The lack of effective disease-modifying therapeutics to tackle Alzheimer's disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of the present study was to investigate the potential long-term protective impact of IHC against AD-related phenotype, putting a special focus on cognition and mitochondrial bioenergetics and dynamics. For this purpose, six-month-old male triple transgenic AD mice (3×Tg-AD) were submitted to an IHC protocol for two weeks and the behavioral assessment was performed at 8.5 months of age, while the sacrifice of mice occurred at nine months of age and their brains were removed for the remaining analyses. Interestingly, IHC was able to prevent anxiety-like behavior and memory and learning deficits and significantly reduced brain cortical levels of amyloid-ß (Aß) in 3×Tg-AD mice. Concerning brain energy metabolism, IHC caused a significant increase in brain cortical levels of glucose and a robust improvement of the mitochondrial bioenergetic profile in 3×Tg-AD mice, as mirrored by the significant increase in mitochondrial membrane potential (ΔΨm) and respiratory control ratio (RCR). Notably, the improvement of mitochondrial bioenergetics seems to result from an adaptative coordination of the distinct but intertwined aspects of the mitochondrial quality control axis. Particularly, our results indicate that IHC favors mitochondrial fusion and promotes mitochondrial biogenesis and transport and mitophagy in the brain cortex of 3×Tg-AD mice. Lastly, IHC also induced a marked reduction in synaptosomal-associated protein 25 kDa (SNAP-25) levels and a significant increase in both glutamate and GABA levels in the brain cortex of 3×Tg-AD mice, suggesting a remodeling of the synaptic microenvironment. Overall, these results demonstrate the effectiveness of the IHC paradigm in forestalling the AD-related phenotype in the 3×Tg-AD mouse model, offering new insights to AD therapy and forcing a rethink concerning the potential value of non-pharmacological interventions in clinical practice.

Structural and functional abnormalities in iron-depleted heart.

Iron deficiency (ID) is a common and ominous comorbidity in heart failure (HF) and predicts worse outcomes, independently of the presence of anaemia. Accumulated data from animal models of systemic ID suggest that ID is associated with several functional and structural abnormalities of the heart. However, the exact role of myocardial iron deficiency irrespective of systemic ID and/or anaemia has been elusive. Recently, several transgenic models of cardiac-specific ID have been developed to investigate the influence of ID on cardiac tissue. In this review, we discuss structural and functional cardiac consequences of ID in these models and summarize data from clinical studies. Moreover, the beneficial effects of intravenous iron supplementation are specified.

[Transgenic mice as models to study the influence of polyunsaturated fatty acids on metabolism]. / Transgeniczne myszy jako model w badaniach wplywu wielonienasyconych kwasów tluszczowych na organizm

The mouse is a popular animal model employed for studying metabolic alterations. The generation of fat-1 transgenic mice by Professor Jing X. Kang and collaborators has revolutionised the omega-3 research. Fat-1 mice are able to convert omega-6 fatty acids to omega-3 fatty acids due to gene fat-1 from Caenorhabditis elegans that encodes an omega-3 fatty acids desaturase. This mice model can endogenously synthesize omega-3 PUFA without ALA intake and the balancing quantity and quality of various confounding factors of different diets. Next, novel transgenic mice - "Omega mice" with the expression of the fat-1 and fat-2 transgenes were created to produce both omega-6 and omega-3 PUFA from a diet containing saturated fat or carbohydrates with essential fatty acids deficiency. Both transgenic mice are utilities for studying molecular mechanisms of omega-3 fatty acids and their metabolites in tumorigenesis and inflammatory disorders.

Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.

High Endogenous Accumulation of ω-3 Polyunsaturated Fatty Acids Protect against Ischemia-Reperfusion Renal Injury through AMPK-Mediated Autophagy in Fat-1 Mice.

Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). Fat-1 transgenic mice produce ω3-Polyunsaturated fatty acids (ω3-PUFAs) from ω6-Polyunsaturated fatty acids (ω6-PUFAs) without a dietary ω3-PUFAs supplement, leading to a high accumulation of omega-3 in various tissues. ω3-PUFAs show protective effects against various renal injuries and it has recently been reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated its associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham (n = 10), fat-1 sham (n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI (n = 15). Kidneys and blood were harvested 24 h after IRI and renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7; lower amounts of p62; and, higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more adenosine monophosphate-activated protein kinase (AMPK) activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.

Application of metabolomics based on direct mass spectrometry analysis for the elucidation of altered metabolic pathways in serum from the APP/PS1 transgenic model of Alzheimer's disease.

Metabolomic analysis of brain tissue from transgenic mouse models of Alzheimer's disease has demonstrated a great potential for the study of pathological mechanisms and the development of new therapies and biomarkers for diagnosis. However, in order to translate these investigations to the clinical practice it is necessary to corroborate these findings in peripheral samples. To this end, this work considers the application of a novel metabolomic platform based on the combination of a two-steps extraction procedure with complementary analysis by direct infusion electrospray mass spectrometry and flow infusion atmospheric pressure photoionization mass spectrometry for a holistic investigation of metabolic abnormalities in serum samples from APP/PS1 mice. A number of metabolites were found to be perturbed in this mouse model, including increased levels of di- and tri-acylglycerols, eicosanoids, inosine, choline and glycerophosphoethanolamine; reduced content of cholesteryl esters, free fatty acids, lysophosphocholines, amino acids, energy-related metabolites, phosphoethanolamine and urea, as well as abnormal distribution of phosphocholines depending on the fatty acid linked to the molecular moiety. This allowed the elucidation of possible pathways disturbed underlying to disease (abnormal homeostasis of phospholipids leading to membrane breakdown, energy-related failures, hyperammonemia and hyperlipidemia, among others), thus demonstrating the utility of peripheral samples to investigate pathology in the APP/PS1 model.

Generation and characterization of transgenic mice with the full-length human DMD gene.

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx x Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx x Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation.

Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin.

To investigate the functional expression of adenosine A3 receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1-2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca2+ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest.

Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response.

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder characterized by extremely elevated levels in plasma homocysteine. Patients homozygous for the I278T or R266K mutations respond clinically to pharmacologic doses of pyridoxine, the precursor of a cofactor for the enzyme, 5'-pyridoxal phosphate (PLP). Here we test the hypothesis that these mutations are pyridoxine responsive because they lower the affinity of the enzyme for PLP. We show that recombinant R266K has 30 to 100% of the specific activity of the wild-type enzyme, while I278T only has only 1 to 5% activity. Kinetic studies show that the decreased activity in both enzymes is due to reduced turnover rate and not substrate binding. Neither I278T nor R266K appear to greatly affect multimer status of the enzyme. The R266K enzyme has reduced affinity for PLP compared to the wild-type enzyme, providing a mechanism for the pyridoxine response observed in patients. Surprisingly, the I278T enzyme does not have altered affinity for PLP. To confirm that this was not an in vitro artifact, we examined pyridoxine response in mice that stably express human I278T as their sole source of CBS activity. These mice have extremely elevated plasma homocysteine levels and do not respond significantly to large doses of pyridoxine. Our findings suggest that there may be multiple mechanisms involved in response to pyridoxine.

MyoR is expressed in nonmyogenic cells and can inhibit their differentiation.

The development of skeletal muscle in mammals is promoted by the muscle-specific basic helix-loop-helix transcription factors of the MyoD family. Evidence also suggests that there are basic helix-loop-helix proteins that specifically inhibit skeletal myogenesis, including Mtwist, Mist1, and the most recently described, MyoR. It has been suggested that MyoR expression is limited to the precursors of the skeletal muscle lineage and acts as a transcriptional repressor of the muscle differentiation program. However, our results demonstrate that MyoR is expressed in several different, nonmuscle adult tissues. Furthermore, MyoR is expressed in the embryonic ectoderm of blastocyst stage mouse embryos, well before skeletal muscle specification and even before delineation of the mesodermal germ layer. Using embryonic ectoderm analogous stem cells, we demonstrate that in these nonmuscle cells, as in skeletal muscle precursor cells, expression of MyoR is inversely correlated with the extent of cellular differentiation as induced by retinoic acid. Our preliminary results indicate that overexpression of exogenous MyoR inhibits retinoic-acid-induced differentiation in EC cells and is lethal to early mouse embryos. Our results suggest a much broader role for MyoR in the repression and/or determination of embryonic cell differentiation.

Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies.

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.

Beneficial effect of ginseng root in SOD-1 (G93A) transgenic mice.

Many patients with amyotrophic lateral sclerosis (ALS; motor neuron disease) use natural or traditional therapies of unproven benefit. One such therapy is ginseng root. However, in some other disease models, ginseng has proven efficacious. Ginseng improves learning and memory in rats, and reduces neuronal death following transient cerebral ischemia. These effects of ginseng have been related to increases in the expression of nerve growth factor and its high affinity receptor in the rat brain, and antioxidant actions, inter alia. Since such actions could be beneficial in ALS as well, we studied the effect of ginseng (Panax quinquefolium), 40 and 80 mg/Kg, in B6SJL-TgN(SOD1-G93A)1Gur transgenic mice. The ginseng was given in drinking water, from age 30d onwards. We measured the time to onset of signs of motor impairment, and survival. There was no difference between the two ginseng groups (n=6, 6) in either measure. However, compared to controls (n=13), there was a prolongation in onset of signs (116d vs. 94d, P<0.001), and survival (139d vs. 132d, P<0.05). These experiments lend support to the use of ginseng root in ALS. Future experiments using this model could examine for symptomatic effects of ginseng, measure the effect of specific ginsenosides (which differ between ginseng species), and elucidate their mechanisms of action.

The 5'-flanking region of the mouse adenylyl cyclase type VIII gene imparts tissue-specific expression in transgenic mice.

The calcium-stimulated adenylyl cyclases (ACs) play a central role in stimulus-dependent modification of synaptic function. The type VIII AC (AC8) is one of three mammalian calcium-stimulated isoforms, each of which is expressed in a region-specific manner in the CNS. To delineate the DNA sequences responsible for appropriate targeting of AC8 expression, we report here the complete structure of the AC8 gene and define the pattern of expression of the full-length cDNA and its splice variants. In addition to expression within the brain, robust expression of AC8 was also found in the lung. By in situ hybridization, we have found the highest expression of AC8 mRNA within the olfactory bulb, thalamus, habenula, cerebral cortex, and hypothalamic supraoptic and paraventricular nuclei. By generating transgenic mice whose expression of beta-galactosidase is controlled by the AC8 5'-flanking DNA sequences, we demonstrate that the DNA sequences within the 10 kb preceding exon 1 are critical for establishment of this region-specific pattern. This spectrum of sites of production is unique to AC8 among the calcium-stimulated adenylyl cyclases and suggests nonredundant functions with other adenylyl cyclases in neuroendocrine regulation and/or behavior.

Effects of immunoneutralization of substance P on hypothalamic neurotransmitters in normal mice and in transgenic mice expressing bovine growth hormone.

It is well known that transgenic mice expressing bovine growth hormone have altered neuroendocrine functions. Substance P was shown to influence the secretion of gonadotropins. In this investigation, the effect of a single injection of an antiserum to substance P was investigated in intact and castrated transgenic (MT-bGH) mice and in their normal litter mates. In the median eminence, the administration of antisubstance P serum resulted in a decreased dihydroxyphenyl acetic acid/dopamine index in intact and castrated normal mice but was without effect in transgenics. The homovanillic/dopamine index was decreased in normal mice (intact or castrated) but unchanged in transgenics. Norepinephrine and epinephrine were increased in normal mice (intact and castrated) treated with the anti-SP serum, but in transgenic mice, the anti-SP serum induced significant changes of norepinephrine only in intact animals, with no modifications in epinephrine levels. In the whole hypothalamus (minus the median eminence), the injection of antisubstance P serum resulted in an increased dihydroxyphenyl acetic acid/dopamine index in castrated, but not in intact, normal mice. In transgenic mice, this index was increased in intact but decreased in castrated animals. The homovanillic/dopamine index was decreased in normal intact mice treated with the antiserum but increased in intact transgenic mice. Norepinephrine and epinephrine were decreased by the antiserum treatment in normal intact mice but were unchanged in transgenics, except for norepinephrine in castrated transgenics, in which it was found increased. The administration of the antiserum did not affect plasma LH, FSH, or prolactin in normal mice but it reduced LH levels in intact transgenic mice. These results indicate that the response to the treatment with the antiserum to substance P shows considerable alterations in transgenic mice as compared with their litter-mate, normal controls, producing divergent effects on hypothalamic catecholamine metabolism. The present findings confirm that transgenic mice overexpressing the bGH gene have marked neuroendocrine alterations as compared with their normal litter mates.

Effect of increased production of growth hormone on body composition in mice: transgenic versus control.

The body composition (water, fat, protein and ash) of male and female transgenic mice which had a sheep metallothionein 1a-sheep growth hormone fusion gene and their non-transgenic controls was determined at intervals from birth to 21 days of age (weaning) in 66 mice of each group, and in an additional 64 mice over the period 25 to 98 days of age. Overall 520 mice were analysed. Weaned mice were starved overnight prior to slaughter. Food was available ad libitum. and, after weaning, a zinc sulphate supplement was added to the drinking water to initiate expression of the transgene. Growth and body composition were similar in all groups before weaning. From 39 days of age, transgenic females became progressively heavier than corresponding controls, being 60% heavier at the end of the experiment. They contained less fat, more water and slightly less ash than did controls of the same live weight but similar amounts of protein. When examined on a fat-free basis, they had less protein and ash and more water than corresponding controls. Appropriate linear and quadratic regression equations are presented to describe the above relationships. Growth and body composition were more variable in transgenic males but, on average, similar to controls.