Somatic signature of brain-specific single nucleotide variations in sporadic alzheimer's disease

© 2014 IOS Press and the authors. All rights reserved. Background: Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimer's disease (SAD), the molecular mechanisms responsible remain largely unknown. Objective: The aim of the study was to investigate the presence of somatic, brain-specific single nucleotide variations (SNV) in the hippocampus of SAD samples. Methods: By using bioinformatic tools, we compared whole exome sequences in paired blood and hippocampal genomic DNAs from 17 SAD patients and from 2 controls and 2 vascular dementia patients. Results: We found a remarkable number of SNVs in SAD brains (~575 per patient) that were not detected in blood. Loci with hippocampus-specific (hs)-SNVs were common to several patients, with 38 genes being present in 6 or more patients out of the 17. While some of these SNVs were in genes previously related to SAD (e.g., CSMD1, LRP2), most hs-SNVs occurred in loci previously unrelated to SAD. The most frequent genes with hs-SNVs were associated with neurotransmission, DNA metabolism, neuronal transport, and muscular function. Interestingly, 19 recurrent hs-SNVs were common to 3 SAD patients. Repetitive loci or hs-SNVs were underrepresented in the hippocampus of control or vascular dementia donors, or in the cerebellum of SAD patients. Conclusion: Our data suggest that adult blood and brain have different DNA genomic variations, and that somatic genetic mosaicism and brain-specific genome reshaping may contribute to SAD pathogenesis and cognitive differences between individuals.BBVA Foundation and MICINN-MINECO. We also like to thank the support of the Reina Sofia Foundation, the CIEN Foundation, CIBERNED (ISCIII

The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2

Producción CientíficaBrain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca2 + -dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates.2015-03-3

Hypoxia and P1 receptor activation regulate the high-affinity concentrative adenosine transporter CNT2 in differentiated neuronal PC12 cells

Under several adverse conditions, such as hypoxia or ischaemia, extracellular levels of adenosine are elevated because of increased energy demands and ATP metabolism. Because extracellular adenosine affects metabolism through G-protein-coupled receptors, its regulation is of high adaptive importance. CNT2 (concentrative nucleoside transporter 2) may play physiological roles beyond nucleoside salvage in brain as it does in other tissues. Even though nucleoside transport in brain has mostly been seen as being of equilibrative-type, in the present study, we prove that the rat phaeochromocytoma cell line PC12 shows a concentrative adenosine transport of CNT2-type when cells are differentiated to a neuronal phenotype by treatment with NGF (nerve growth factor). Differentiation of PC12 cells was also associated with the up-regulation of adenosine A1 receptors. Addition of adenosine receptor agonists to cell cultures increased CNT2-related activity by a mechanism consistent with A1 and A2A receptor activation. The addition of adenosine to the culture medium also induced the phosphorylation of the intracellular regulatory kinase AMPK (AMP-activated protein kinase), with this effect being dependent upon adenosine transport. CNT2-related activity of differentiated PC12 cells was also dramatically down-regulated under hypoxic conditions. Interestingly, the analysis of nucleoside transporter expression after experimental focal ischaemia in rat brain showed that CNT2 expression was down-regulated in the infarcted tissue, with this effect somehow being restricted to other adenosine transporter proteins such as CNT3 and ENT1 (equilibrative nucleoside transporter 1). In summary, CNT2 is likely tomodulate extracellular adenosine and cell energy balance in neuronal tissue. © 2013 Biochemical Society.Peer Reviewe

The Wnt/Frizzled pathway induces the degradation of Alex3 protein.

<p>(<b>A</b>) WBs showing that Wnt1 co-transfection (left) and incubation with Wnt1-conditioned media (CM, right), but not treatment with Wnt3a (200 ng/ml) (middle), induces the degradation of Alex3 protein. (<b>B</b>) WB showing that co-transfection with Wnt1, Fz2, Wnt5a and Wnt11 lead to different reductions in Alex3 protein levels (left). Recombinant Wnt5a also induces Alex3 degradation in a concentration-dependent manner (right).</p

The N-terminal domain of Alex3 is sufficient to induce mitochondrial aggregation.

<p>(<b>A–D</b>) Overexpression of Alex3-GFP (green) in HEK293T cells induces severe alterations of the mitochondrial network when compared with the expression of control GFP (<b>A</b>). (<b>B</b>) Illustrates an Alex3-transfected cell displaying normal mitochondrial morphology; (<b>C,D</b>) Alex3-overexpressing cells showing mild aggregating phenotypes (<b>C</b>) and severe aggregating mitochondrial phenotypes (<b>D</b>); Alex3 protein was visualized in green, mitochondria in red (MitDsRed), and nuclei were labeled with bisbenzimide (blue). (<b>E</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in control (GFP) and Alex3-GFP-overexpressing cells. (<b>F</b>) Top: Scheme of the Alex3-GFP deletion constructs used for transfection. Bottom: Western Blot showing representative truncated Alex3-GFP constructs at the predicted protein sizes. (<b>G–J</b>) Photomicrographs illustrating that expression of the Alex3(1–200)-GFP (<b>G</b>), Alex3(1–106)-GFP (<b>H</b>) and Alex3(1–30)-GFP (<b>I</b>) constructs leads to mitochondrial aggregation; in contrast, deletion of the first N terminal 12 aa (GFP-Alex3ΔNt) targets Alex3 protein to the nucleus (<b>J</b>). Note that the 30 aa N-terminus deletion construct has a truncated outer mitochondrial membrane localization sequence, which may interfere with its mitochondrial targeting, thereby leading to nuclear localization. (<b>K</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293T cells after transfection with several truncated Alex3-GFP constructs; the data show that all the constructs containing the N terminal region cause mitochondrial aggregation. Alex3 protein was visualized in green (GFP), mitochondria in red (MitDsRed) and nuclei in blue (bisbenzimide). Scale bar: 10 µm.</p

Alex3 degradation by Wnt1 is independent of the proteasome, JNK, CAMKII and Calcineurin pathways.

<p>(<b>A</b>) Proteasome inhibition with 10 µM MG-132 treatment blocks the normal turnover of Alex3 protein but not its Wnt1-induced degradation. (<b>B</b>) Numerous Alex3-overexpressing HEK293AD cells treated with the proteasomal inhibitor MG132 show the most severe mitochondrial aggregating phenotype. (<b>C</b>) Inhibition of JNK with 10 µM SP600125 (downstream effector of the Wnt/PCP pathway), CAMKII with 25 µM KN62 or Calcineurin with 10 µM Cypermetrin (downstream effectors of the Wnt/Ca²⁺ pathway) do not induce Alex3 protein degradation. Scale bar: 10 µm.</p

PKC and CKII phosphorylation protects against Wnt/Frizzled degradation of Alex3.

<p>(<b>A</b>) Inhibition of CKII (with 100 µM casein kinase II inhibitor I), downstream effector of the Wnt signaling pathway, is sufficient to trigger Alex3 degradation. (<b>B</b>) In contrast, PKC activation with 1 µM TPA protects against Wnt1-induced degradation of Alex3 protein. (<b>C</b>,<b>D</b>) Inhibition of PKC (with 1 µM Calphostin C) and treatment with 20 µM BAPTA/AM, an intracellular calcium chelator, also reproduces Wnt1 degradation. (<b>E</b>) Photomicrographs demonstrating that treatment with TPA prevents Alex3 degradation induced by Wnt1 and the reversion to normal mitochondrial phenotypes. (<b>F</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293AD cells in the conditions shown in (<b>E</b>); note that incubation with TPA prevents the rescue of mitochondrial phenotypes induced by Wnt1. Scale bar: 10 µm. The quantification of Alex3 protein levels is shown at the bottom.</p

Schematic representation of Alex3 protein.

<p>Predicted domains are annotated on the basis of databases such as Pfam, Smart or Wolfpsort and bibliographic references. The stars show the position of putative phosphorylation sites in serine or threonine residues by CK2, PKC and PKA kinases.</p

Alex3 degradation is independent of the canonical Wnt/β-catenin pathway.

<p>(<b>A,B</b>) Constitutively active β-catenin (red) neither induces Alex3 protein degradation, as seen in WB (<b>A</b>), nor reverts the aggregated mitochondrial phenotypes induced by Alex3 overexpression (green in <b>B</b>). Nuclei were visualized in blue (bisbenzimide) (<b>B</b>). (<b>C,D</b>) Neither co-transfection with Dvl2 (<b>C</b>) nor the inhibition of GSK3β with 10 mM LiCl or with 10 µM SB212763 (<b>D</b>) induces Alex3 protein degradation. Wnt1 transfection was used as a control for Alex3 degradation. Scale bar: 10 µm.</p

Wnt1 increases mitochondrial motility and dynamics.

<p>Series of representative confocal images, taken every 225 sec, from live HEK293T cells overexpressing the mitochondrial tagged protein MitDsRed (<b>A</b>)<b>,</b> Alex3-GFP fusion protein (<b>B</b>), or Alex3-GFP and Wnt1 cDNAs (4∶1) (<b>C,D</b>). In (<b>D</b>) TPA treatment was used to activate PKC. Arrows identify areas with highly dynamic mitochondria. While mitochondrial motility is high in control (<b>A</b>) and Alex3-GFP/Wnt1 (<b>C</b>) conditions, it is severely reduced in Alex3-GFP-overexpressing cells (<b>B</b>) and in Alex3-GFP/Wnt1/TPA-treated cells (<b>D</b>). (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067773#pone.0067773.s008" target="_blank">Videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067773#pone.0067773.s009" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067773#pone.0067773.s010" target="_blank">S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067773#pone.0067773.s011" target="_blank">S4</a>). Scale bar: 10 µm.</p