Expression of NHEs inhibits formation of mutant HTT aggregates.

<p>Representative photomicrographs of Neuro-2a cells overexpressing plasmids for indicated protein (A), and quantification of the aggregate-positive cell percentage (B). Insets show extranuclear and intranuclear localization of polyQ aggregates by expression of NES-81Q and NLS-81Q, respectively. Scale bar = 30 µm in (A).</p

NHE5 null mutation increases polyglutamine protein aggregation in vivo.

<p>Representative photomicrographs of polyglutamine protein inclusions formed in cerebellar cortex of mice of the indicated genotype are shown in A. Immunoreactivity of HTT, p62, and DAPI staining images are shown with high-manification view of granular layer inclusions in insets. Scale bar = 30 µm. Quantification of the diameter (B) and number (C) of the aggregates are shown. See Method section for details. In B, boxes represent lower quartile and upper quartile, with bars showing 1.5× of the lower and upper quartile values. White and black circles show max and min, respectively, and quadrangles show median data points. *P<0.001 (Mann-Whitney's U-test). In C, data shown are mean ± SD. *P<0.05(Student's t-test).</p

Acidification by polyglutamine protein expression visualized by dKeima-mem-based subcellular pH imaging.

<p>A. Representative subcellular pH imaging data in Neuro-2a cells expressing dKeima-mem during acidification followed by recovery in Na⁺-free and Na⁺-containing medium. B. Expression of NHE1 and NHE5 but not by their non-functional mutants facilitates recovery from acidification (see Methods). Mean pH recovery rate ±SD for each type of cells is shown as a bar graph. *P<0.01, **P<0.05 (Student's t-test; in comparison with the recovery rate of vector-only transfected control cells). Numbers in parentheses on X axis indicates the number of cells examined. C, D. Representative intracellular pH imaging results of Neuro-2a cells expressing dKeima-mem together with HttEx1-Q25 or HttEx1-Q97 demonstrated by pseudo-color images (C) and box whisker plot of quantification data 445 nm/586 nm emission ratio(D). In D, boxes represent lower quartile and upper quartile, with bars showing 1.5× of the lower and upper quartile values. White and black circles show max and min, respectively, and quadrangles show median data points. *P<0.001 (Welch's t test).</p

Neuro-2a cells show increased autophagosome formation in response to environmental pH modulation.

<p>A, B Representative photomicrographs of Neuro-2a cells stably expressing EGFP-LC3 maintained under indicated medium and atmosphere conditions for 2 hr (A), or with indicated reagents in serum-containing DMEM under 5% CO2 (B). Note the significantly increased autophagosome formation in the cells maintained in Krebs-Ringer bicarbonate buffer (KRB), which contains electrolites and glucose only under 100% normal air, and with EIPA. C Representative images of immunoblot analysis for expression of p62 and LC3 in EGFP-LC3-expressing Neuro-2a cells with indicated reagents. Bar graphs in C show quantification data of LC3-II expression levels relative to the level in EGFP-LC3-expressing Neuro-2a cells with no additional treatment normalized to β-actin expression. Results shown are mean ± SEM. *P<0.01, **P<0.05 (Student's t- test). Note that EIPA-treated cells show increased p62 and LC3-II levels indicating the inhibition of autophagic degradation by NHE inhibition. D. Detection of NHE family members in Neuro-2a cells by RT-PCR.</p

Induction of autophagy in Neuro-2a cells by NHE1 and NHE5.

<p>Representative photomicrographs of Neuro-2a cells stably expressing EGFP-LC3 with additional expression of NHE1 or NHE5 (A). Scale bar = 50 µm. Representative images of immunoblot analysis for expression of p62 and LC3 in EGFP-LC3-expressing Neuro-2a cells overexpressing indicated constructs (B). Bar graphs in B show quantification data of LC3-II expression levels relative to the level in EGFP-LC3-expressing Neuro-2a cells with no additional treatment normalized to β-actin expression. Results shown are mean ± SEM. *P<0.01, **P<0.05 (Student's t- test). Note that induced levels of LC3-II by overexpression of NHE1 and NHE5 was further increased by E64d/PepA, suggesting that expression of NHE1 and NHE5 induces autophagy in B.</p

Biological <i>in Vitro</i> and <i>in Vivo</i> Studies of a Series of New Asymmetrical Cationic [^99mTc(N)(DTC-Ln)(PNP)]⁺ Complex (DTC-Ln = Alicyclic Dithiocarbamate and PNP = Diphosphinoamine)

99mTc(N)-DBODC5 is a cationic mixed compound under clinical investigation as potential myocardial imaging agent. In spite of this, analogously to the other cationic 99mTc-agents, presents a relatively low first-pass extraction. Thus, modification of 99mTc(N)-DBODC(5) direct to increase its first-pass extraction keeping unaltered the favorable imaging properties would be desirable. This work describes the synthesis and biological evaluation of a series of novel cationic 99mTc-nitrido complexes, of general formula [99mTcN(DTC-Ln)(PNP)]+ (DTC-Ln= alicyclic dithiocarbamates; PNP = diphosphinoamine), as potential radiotracers for myocardial perfusion imaging. The synthesis of cationic 99mTc-(N)-complexes were accomplished in two steps. Biodistribution studies were performed in rats and compared with the distribution profiles of 99mTc(N)-DBODC5 and 99mTc-Sestamibi. The metabolisms of the most promising compounds were evaluated by HPLC methods. Biological studies revealed that most of the complexes have a high initial and persistent heart uptake with rapid clearance from nontarget tissues. Among tested compounds, 2 and 12 showed improved heart uptake with respect to the gold standard 99mTc-complexes with favorable heart-to-liver and slightly lower heart-to-lung ratios. Chromatographic profiles of 99mTc(N)-radioactivity extracted from tissues and fluids were coincident with the native compound evidencing remarkable in vivo stability of these agents. This study shows that the incorporation of alicyclic dithiocarbamate in the [99mTc(N)(PNP)]+ building block yields to a significant increase of the heart uptake at early injection point suggesting that the first-pass extraction fraction of these novel complexes may be increased with respect to the other cationic 99mTc-agents keeping almost unaltered the favorable target/nontarget ratios