CaM/Tet-DT_A mice develop neuronal loss in Alzheimer-disease related brain areas.

<p>A 25-day withholding of doxycycline from the diet of CaM/Tet-DT_A mice induces DT_A expression, which causes a selective loss of hippocampal and cortical neurons. Representative images depict a significant loss of NeuN immunoreactivity in the CA1 and DG of the hippocampus (<b>A</b>), and layers II/III of the entorhinal cortex (<b>B</b>, <i>n</i> = 5–6, quantified by optical densitometry in <b>C</b>). Crossing CaM/Tet-DT_A mice with mice carrying a Thy1-GFP-M transgene produces sparse GFP-labeling of CA1 pyramidal neurons (<b>D</b>). Scale bars = 1 mm.</p

7,8-DHF treatment improves spatial learning and memory in CaM/Tet-DT_A mice.

<p>Lesioned CaM/Tet-DT_A mice exhibit a significantly increased preference for the open arms of an elevated plus maze compared to controls (<b>A</b>, <i>n</i> = 9–12, <i>p</i><0.0001). All groups made a similar total number of arm entries (<b>B</b>). 7,8-DHF treatment did not alter preference for open or closed arms (<b>A–B</b>). A rotarod test reveals no differences between groups in the time to fall off an accelerating rod (<b>C</b>). During water maze training, lesioned mice treated with 7,8-DHF were not significantly slower to reach the escape platform compared to controls, except for day 3 (<b>D</b>, <i>n</i> = 9–12, <i>p</i><0.001). On day 4, lesioned mice treated with 7,8-DHF reached the platform significantly faster than lesioned mice that received vehicle (<b>D</b>, <i>n</i> = 9–12, #, <i>p</i><0.05). On the 24 hr probe trial, latencies to reach and mean proximity to the former platform location were not significantly different between 7,8-DHF-treated lesioned mice and controls (<b>E–F</b>, <i>n</i> = 9–12, <i>p</i>>0.05). All groups had a similar average swim speed during the probe trial (<b>G</b>).</p

7,8-DHF treatment increases thin spine density on dendrites in CA1 pyramidal neurons.

<p>High-magnification confocal imaging and analysis of dendrites from GFP-expressing CA1 pyramidal neurons reveals elevated total spine density in 7,8-DHF-treated lesioned mice (<b>A–B</b>, <i>n</i> = 5–7, <i>p</i><0.05). Classification of thin (T), mushroom (M), and stubby (S) spines indicates a specific elevation of thin spines in lesioned mice (<b>C</b>, <i>n</i> = 5–7, <i>p</i><0.05), which is further elevated by 7,8-DHF treatment (<i>n</i> = 5–7, <i>p</i><0.01 versus vehicle-treated lesioned mice). Analysis of synaptophysin immunoreactivity reveals a lesion-induced increase in presynaptic terminal packing density in CA1 stratum oriens (ori) and radiatum (rad) (<b>D–E</b>, <i>n</i> = 5, <i>p</i><0.05), and loss of density in the stratum lacunosum-moleculare (<b>D–E</b>, <i>n</i> = 5, <i>p</i><0.01), but no effect of 7,8-DHF. Labeling for MAP2 reveals a significant loss of CA1 pyramidal neuron dendrites in lesioned groups (<b>F–G</b>, <i>n</i> = 5–7, <i>p</i><0.001), and no impact of 7,8-DHF treatment on dendrite density. Scale bar = 1 µm.</p

<i>mei-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>mei-1(or642 </i>ts<i>) and mei-1(or646 </i>ts<i>)</i> embryos. In the <i>mei-1</i> mutants the polar bodies were large and misshapen and embryos contained multiple [top <i>mei-1(or642 </i>ts<i>)</i> embryo and <i>mei-1(or646 </i>ts<i>)</i>] or zero maternal pronuclei (second <i>mei-1(or642 </i>ts<i>)</i> embryo). The two <i>mei-1(or642 </i>ts<i>)</i> embryos were obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes, the <i>mei-1(or646 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 7 hours prior to imaging. White arrowheads indicates polar bodies, black arrowheads indicate multiple maternal pronuclei, the black arrow denotes multiple nuclei per cell at the two cell stage, and the “p” refers to the paternal pronucleus in an embryo lacking a maternal pronucleus. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Defect maps of individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal polar body size, 2; normal pronuclear number, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes. C. Amino acid alteration in the mutants. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

<i>zyg-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or297 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos. In the <i>zyg-1</i> mutants the two cell stage blastomeres assembled monopolar spindles, cytokinesis failed, and there were multiple nuclei present at the four cell equivilent stage. The <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The <i>zyg-1(or297 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes prior to imaging. Black arrows indicate normal bipolar spindles in the wild-type embryo and white arrowheads indicate multiple nuclei present at the four cell equivalent stage. Times in min:sec are given relative to AB nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Amino acid alterations in the mutants. Asterisks indicate the changed residues. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect maps for the <i>zyg-1</i> mutants.Individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal two cell embryo, 2; bipolar spindles at two cell stage, 3; one nucleus per cell at four cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes.</p

The phenotypes of the TS mutants when grown at the restrictive temperature from the L1 larval stage.

1<p>We determined the L1 upshift phenotype by plating hypochlorite-synchronized L1 larvae and incubating them at 26°C until they reached adulthood. Abbreviations: Egl: egg laying-defective, Emb: embryonic lethal, Pvl: protruding vulva, Sb: small broods, Ste: sterile.</p

<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

Summary of the TS mutant loci and comparison of previously available alleles.

1<p>Information obtained from: <a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>.</p

Determination if the TS mutations are potentially fast-acting.

1<p>We determined if an allele was potentially fast-acting in the following manner: We mounted embryos produced at 15°C on microscope slides and immediately made time-lapse videomicrographs at a room maintained at 24°C. If defects similar to those observed after long temperature shifts were found in at least 20% of the embryos and if there was little embryonic lethality at 15°C, we conclude that the allele may be fast-acting. We have labeled these cases as “Yes”. However, if there was significant embryonic lethality at 15°C, we cannot conclude that the presence of cellular defects after short upshifts is due to the upshift or to defects that occur even at 15°C. We have labeled these cases as “Unclear”.</p>2<p>For <i>mei-1, par-2,</i> and <i>zyg-1</i>, we incubated mutant worms at 26°C for 30 minutes prior to imaging (instead of the usual∼1 min. upshift) because the gene products appeared to be required prior to when we started our imaging (pronuclear migration).</p>3<p>High lethality at the permissive temperature precludes making a determination.</p>4<p>The low penetrance of severe defects precludes making a determination.</p

A <i>plk-1</i> mutant.

<p>A. DIC time-lapse images of wild-type and <i>plk-1(or683 </i>ts<i>)</i> embryos. In the <i>plk-1</i> mutant the nuclear centrosomal complex (NCC) failed to rotate, a transverse P₀ spindle assembled, and the daughter blastomeres were binucleate. The <i>plk-1(or683 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 6 hours prior to imaging. Black dots represent centrosomes/spindle poles and asterisks denote multiple nuclei per cell at the two cell stage. Times in min:sec are given relative to NEBD. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; nuclear centrosomal complex rotation, 2; spindle alignment, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.</p