hAPP₆₉₅ (hAPP), but not hAPP-swe, effectively rescued defects in axonal outgrowth in APPb-MO morphants.

<p>(A) Co-injection of hAPP₆₉₅ mRNA rescued APPb morphant embryonic morphology. As depicted, embryos were inspected over a period of 3 days. Co-injection of full length human APP₆₉₅ mRNA rescued the defective phenotype observed in the APPb morphant embryos (*, p = 0.026, p<0.05 in two tailed paired <i>t-test</i>). In contrast, human APP-Swedish mutation (Δ, p = 0.5241, p>0.05) and AICD mRNA failed to rescue the APPb morphant phenotype. Combined injections (10 ng of APPb-MO and 350 pg of AICD mRNA) caused more severe deficits compared to injecting APPb-MO alone. (B) Co-injection of hAPP₆₉₅ mRNA rescued axonal outgrowth of motor neurons Vp and VII of the APPb morphant. Zebrafish embryos co-injected with full length human APP₆₉₅ rescued the APPb morphant axonal outgrowth phenotype of motor neurons Vp and VII (arrow). (C) Embryos co-injected with hAPP₆₉₅ rescued the defective phenotype of motor axons in the spinal cord in APPb morphant embryos. Uninjected embryos expressed normal motor neuron projections from the spinal cord to the myotomes (5–10 somites). Embryos injected with (10 ng) APPb-MO expressed severe motor neuron axonal defects including aberrant projections and decreased branching. Embryos co-injected with APPb-MO and hAPP₆₉₅ mRNA had a rescued phenotype compared to the morphant group. Lateral views of 3 dpf embryos (anterior is to the left, dorsal is at the top). (D) Quantification of embryos expressing normal axonal outgrowth of neurons Vp and VII and spinal cord rescued through co-injection of hAPP695 mRNA. Injection of 10 ng of APPb-MO caused abnormal axonal outgrowth of motor neurons Vp and VII in 75% of embryos. Co-injection with mRNA encoding full-length human APP695 overwhelmingly rescued the APPb morphant phenotype (p = 0.0075, p<0.05 in two tailed paired <i>t</i>-test) (3 dpf). Embryos injected with 10 ng APPb-MO showed significant inhibition of spinal cord axon outgrowth. Embryos co-injected with 10 ng of APPb-MO and 350 pg of hAPP695 mRNA rescued the APPb morphant phenotype (p = 0.0101, p<0.05 in two tailed paired <i>t</i>-test).</p

APPb is required for normal zebrafish embryonic development.

<p>(A) Schematic representation of APPb-MO blocking the mRNA splicing site between intron 1 and exon 2 (indicted in red) as used in this study. (B) Western blot analysis of APPb protein in zebrafish embryos at 2 dpf. The lower panel was probed with anti-GAPDH antibody. At 2 dpf, APPb protein migrated as a doublet at 98 KDa with stronger expressions in the un-injected group and control MO group. With the injection of 10 ng of APPb MO per embryo, APPb protein levels were not detected at 2 dpf. With the injection of 4 ng of APPb MO per embryo, weak expression of APPb protein migrating at 98 KDa was observed. (C) Morphological features of control embryos (a, d, g) and APPb morphant embryos (b, c, e, f, h). Lateral views (anterior to the left and dorsal at the top) of zebrafish embryos. The gross anatomical phenotype included a deformed body and a shortened and curved tail. In addition, defects in midbrain patterning were observed (arrows). 1 dpf: a, b, c; 2 dpf: d, e, f; 3 dpf: g, h. (D) APPb mRNA rescues the defective phenotype. There is little effect on normal embryonic development caused by the injection of control morpholino (APPb mis-match MO). Zebrafish embryos injected with 10 ng of APPb-MO expressed abnormal phenotypes at the 1 dpf (red), 2 dpf (blue) and 3 dpf (yellow) developmental stages. Embryos co-injected with 10 ng of APPb-MO and 350 pg APPb mRNA expressed normal phenotypes during embryogenesis. Statistical significance was measured comparing APPb-MO embryos and the co-injected embryos (p = 0.016, p<0.05, in 2-tailed paired <i>t</i> tests).</p

Neuronal cell cultures derived from embryos injected with APPb-MO had decreased neurite length.

<p>(A) Neuronal culture from control embryos and APPb mRNA (350 pg) over-expression embryos exhibited on average 3 primary branches with several secondary branches. Neuronal cultures from embryos injected with 8 ng of APPb-MO exhibited on average 1 primary branch with many secondary branches. 8 ng of APPb-MO is sufficient to inhibit neruite growth, so embryos were injected with 8 ng of the APPb MO instead of the 10 ng to limit the potential for toxicity. The neurons were cultured for 2 days before fixing. (B) Down-regulation of APPb in cultured neurons affected normal neurite growth. The neurons from the embryos injected with 8 ng of APPb-MO showed a decrease in neurite length compared to neurons from the control embryos (un-injected). The average neurite length of a control neuron was about 130 µm, compared to only about 100 µm in the morphant embryos. Statistical significance was observed between control neurons and APPb knockdown neurons (8 ng APPb MO) (p = 0.0007, p<0.05 in two-tailed paired <i>t-test</i>). (C) Quantifying the effects of APPb mRNA in cultured neurons. No significant difference was observed in neurite length between control and APPb mRNA over-expression cultured neurons. Control cultured neurons expressed a shorter neurite length compared to APPb mRNA over-expression neurons. No statistical significance was observed (p = 0.1915, p>0.05 in two tailed paired <i>t-test</i>). (D) There were fewer branches of neurites in the APPb knockdown neurons (8 ng of APPb MO) than in control neurons (WT); only branches with more than 5 µm were counted. The neurons were cultured for 2 days. (E) Down-regulation of APPb in cultured neurons affected the number of branch tips. Compared to control neurons, APPb-MO cultured neurons showed a decreased number of branch tips of the longest neurite. Statistical significance was observed between control and APPb-MO cultured neurons (p = 0.0074, p<0.05 in two tailed paired <i>t-test</i>). (F) Down-regulation of APPb in cultured neurons affected the morphology and projection of growth cones and filopodia. APPb knockdown neurons expressed abnormal growth cone morphology and a decreased number of filopodia (20 hour neuron cultures). (G) Reduced number of filopodia in APPb knockdown cultured neurons. Compared to control cultured neurons, APPb-MO (8 ng per embryo) cultured neurons expressed a reduced number of filopodia. Statistical significance was observed between control and APPb cultured neurons (p = 0.0091, p<0.05 in two tailed paired <i>t-test</i>).</p

Embryos co-injected with APPb-MO and APPb mRNA rescued defective phenotypes of the Vp and VII and spinal cord nerve projections observed in APPb morphants.

<p>(A) Knockdown of APPb disrupts the projections of axons Vp and VII (3 dpf) when injected with APPb MO. Uninjected embryos and embryos injected with control MO did not show altered axonal outgrowth of neurons Vp and VII. Embryos injected with APPb-MO expressed axonal inhibition of axons Vp and VII (arrows). The defective phenotype was rescued by co-injection of APPb-MO and APPb mRNA. Ventral view; anterior, top. (B) Quantification of embryos expressing normal axonal outgrowth of neurons Vp and VII rescued through co-injection of APPb-MO (splice-block) and APPb mRNA. Uninjected embryos and embryos injected with control MO showed normal axonal growth of Vp and VII neurons. Embryos co-injected with 10 ng of APPb-MO and 350 pg APPb mRNA rescued the defected phenotype observed in the APPb morphant group. Statistical significance was established between APPb-MO embryos and co-injected embryos (p = 0.0168, p<0.05, in 2-tailed paired <i>t</i> test). (C) The translation-block MO of the APPb caused the identical defected phenotype on the axonal outgrowth of the Vp & VII neurons as the splice-block MO of the APPb; both were dose-dependent. (D) The morphants showed an identical defected phenotype on axonal outgrowth of Vp & VII neurons when co-injected with the translation-block MO (3 ng per embryo) of the APPb and the splice-block MO (6.5 ng per embryo) of the APPb at lower doses that did not produce a defective phenotype individually. (E) There was no defective phenotype of axonal outgrowth of Vp & VII neurons in the APPa morphants when injected with the translation-block MO against APPa. (F) APPb function is required for normal nerve outgrowth of the spinal cord. Lateral views of 3 dpf embryos (anterior is to the left, dorsal is at the top). Uninjected embryos and control embryos expressed normal motor nerve projections from the spinal cord to the myotomes (5–10 somites). Embryos injected with 10 ng of APPb-MO (splice-block) expressed severe motor neuron axon defects, including aberrant projections and decreased branching (arrows pointing at axon). Embryos co-injected with APPb-MO and APPb mRNA rescued the severe phenotype observed in the morphant group. The white rectangle in (c) is an amplification of the spinal cord neurons, which shows branching defects of the neurites in the APPb morphants. (G) Downregulation of APPb affects normal projection of motor nerves in the spinal cord. Compared with the control MO and uninjected groups, embryos injected with 10 ng APPb-MO (splice-block) showed significant axonal inhibition. Embryos co-injected with 10 ng of APPb-MO and 350 pg of APPb mRNA rescued the APPb morphant phenotype. Statistical significance was observed between morphant embryos and co-injected embryos (p = 0.0001, p<0.05 in 2-tailed paired <i>t</i> test).</p

Analysis of Transmission Electron Microscopy images of axons in zebrafish hindbrain and trunk regions (5 dpf).

<p>(A) Transmission Electron Microscopy (TEM) images of axons in zebrafish hindbrain. Compared to uninjected and control MO-injected embryos, zebrafish embryos injected with APPb-MO (8 ng) expressed a decreased density and disorganization of the cytoskeleton in both the Mauthner (M) axons and the axons around the M axon. Panels b, d, and f are amplifications of the boxed areas in panels a, c and e, respectively. The white rectangles in panels b, d, and f are amplifications of the areas marked with red 5-point stars. (B) Quantifying the defects of axonal cytoskeletal morphology in the hindbrain of APPb morphant embryos. At 5 dpf, zebrafish embryos injected with 8 ng of APPb-MO showed a disruption in axon cytoskeletal dynamics in the hindbrain region. For the TEM experiment, the embryos were injected with 8 ng of the APPb MO instead of 10 ng. The embryos that were injected with 8 ng of the APPb MO experienced disorganization of the axonal cytoskeleton; the cytoskeleton of the embryos that were injected with 10 ng of APPb MO experienced severely defects in or loss of axons. Statistical significance was observed comparing uninjected (263 axons) and morphant (662 axons) larvae (p = 0.0029, p<0,05 in two-tailed paired <i>t-test</i>). Statistical significance was also observed between control MO (217 axons) and morphant larvae (p = 0.0466, p<0.05 in two tailed paired <i>t-test</i>). (C) TEM images of axons at in the zebrafish trunk section. Compared to uninjected and control MO-injected larvae, zebrafish larvae injected with 8 ng of APPb-MO expressed a decrease in axonal density and had defects in cytoskeletal organization of axons, including the M axon. Panels b, d, and f are amplifications of the boxed areas in panels a, c and e, respectively. The white rectangles in panels b, d and f are amplifications of the areas marked with red 5-point stars. (D) Quantifying defects of axonal cytoskeletal morphology in the trunk region of APPb morphant embryos. At 5 dpf, zebrafish embryos injected with 8 ng of APPb MO expressed an abnormal phenotype in axonal cytoskeletal organization in the trunk region. Statistical significance was observed comparing uninjected (217 axons) and morphant (562 axons) larvae (p = 0.0006, p<0.05 in two tailed paired <i>t-test</i>). Statistical significance was also observed between control MO (212 axons) and morphant larvae (p = 0.0016, p<0.05 in two tailed paired <i>t-test</i>).</p

Tau Protein Mediates APP Intracellular Domain (AICD)-Induced Alzheimer’s-Like Pathological Features in Mice

<div><p>Amyloid precursor protein (APP) is cleaved by gamma-secretase to simultaneously generate amyloid beta (Aβ) and APP Intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer’s disease (AD) pathogenesis but recent studies suggest that amyloid-independent mechanisms also contribute to the disease. We previously showed that AICD transgenic mice (AICD-Tg) exhibit AD-like features such as tau pathology, aberrant neuronal activity, memory deficits and neurodegeneration in an age-dependent manner. Since AD is a tauopathy and tau has been shown to mediate Aβ–induced toxicity, we examined the role of tau in AICD-induced pathological features. We report that ablating endogenous tau protects AICD-Tg mice from deficits in adult neurogenesis, seizure severity, short-term memory deficits and neurodegeneration. Deletion of tau restored abnormal phosphorylation of NMDA receptors, which is likely to underlie hyperexcitability and associated excitotoxicity in AICD-Tg mice. Conversely, overexpression of wild-type human tau aggravated receptor phosphorylation, impaired adult neurogenesis, memory deficits and neurodegeneration. Our findings show that tau is essential for mediating the deleterious effects of AICD. Since tau also mediates Aβ-induced toxic effects, our findings suggest that tau is a common downstream factor in both amyloid-dependent and–independent pathogenic mechanisms and therefore could be a more effective drug target for therapeutic intervention in AD.</p></div

Tau mediates AICD-induced AD-like phenotypes.

<p>(A, B) AICD activates GSK-3β; this in turn phosphorylates tau, which accumulates in somato-dendritic compartments. Increased somato-dendritic tau results in a number of AD-like features in AICD animals, including phosphorylation of NMDA receptors (NMDAR), which leads to increased susceptibility to excitotoxicity. (C) Increasing the amount of tau by transgenic overexpression of human tau (h-Tau) resulted in increased somato-dendritic tau and elevated phosphorylation of NMDAR. Increased NMDAR phosphorylation led to increased susceptibility to excitotoxic effects and aggravated AD-like pathologies. (D) Loss of tau prevented increased NMDAR-phosphorylation, resulting in normalization/lowering of hyper-excitability, finally reducing/ameliorating several key AD-like features in AICD mice.</p

Deficiency of tau protects against age-dependent neurodegeneration and memory loss measured by deficits in spontaneous alternations in AICD-Tg mice.

<p>A. AICD-Tg mice exhibit decreased working memory as measured by deficits in spontaneous alternations at older ages. At >18 months of age, AICD-Tg mice showed decreased spontaneous alternations (working memory) compared to age-matched wild-type animals. Lack of tau (Tau-/-) was able to protect AICD-Tg mice from such deficits in working memory. B. Normal exploratory behavior for all the genotypes measured in the Y-maze. n = 10–12 mice per group for behavioral experiments (A and B). C. AICD-Tg mice at more than 18 months of age showed neuronal loss as assessed by NeuN staining in the CA3 region. Arrows point to focal loss of neurons. Genetic ablation of tau (Tau-/-) protected against neurodegeneration in AICD-Tg mice. D. Quantification of NeuN-positive nuclei. n = 4 for all groups. *p<0.05, **p<0.01 one way ANOVA (Mean ± SEM). Scale bar = 50 μm. WT = wild-type.</p

Tau overload causes behavioral deficits and maintains neurodegeneration at older ages.

<p>A. hTau animals also showed decreased working memory as measured by deficits in spontaneous alternations compared to age-matched wild-type animals. AICD-Tg mice exhibit decreased working memory at older ages compared to age-matched wild-type animals. B. Evidence of normal exploratory behavior was demonstrated for all the genotypes measured in the Y-maze. n = 10–12 mice per group for behavioral experiments (A and B). C. Old AICD-Tg mice carrying an overload of human tau showed neuronal loss similar to age-matched AICD-Tg mice without human tau as assessed by NeuN staining in the CA3 region. Arrows point to focal loss of neurons. Though not statistically significant, there was a trend toward hTau mice having less neuronal cell bodies in the CA3 compared to age-matched wild-type animals. D. Quantification of NeuN-positive nuclei. n = 5 for all. *p<0.05, **p<0.01, ***p<0.001 one way ANOVA (Mean ± SEM). Scale bar = 50 μm</p

Tau knockout prevents sensitivity to Kainic acid–mediated stress in AICD-Tg mice.

<p>A. AICD-Tg mice exhibit increased sensitivity towards Kainic Acid (KA)-mediated seizures at 3–4 months of age. Animals were injected with a sub-threshold dose of KA (20 mg/kg) and observed for 60 minutes for seizure–like hyperactivity. Seizures were scored according to a modified Racine scale. AICD-Tg mice began to show significant signs of seizures at 30 min. However, lack of tau (Tau-/-) significantly protected AICD-Tg mice against KA-mediated seizure-like hyperactivity. Tau-/- mice behaved similar to wild-type (WT) littermates. B. The time taken to reach level 4–5 seizures was scored as the latency to reach convulsive seizures. Lack of tau (Tau-/-) had a protective effect on AICD transgenic animals. C. Mean seizure severity scores showed a significant rescue of seizure severity by Tau-/- in AICD animals. n = 4–5 mice per group (A-C). D. Sagittal sections from 4-month-old WT and AICD-Tg mice were stained with NeuN antibody. Arrows show the loss of neurons in the CA3 region of AICD-Tg mice. Deletion of Tau protected AICD-Tg mice from KA-mediated neuronal loss. E. Quantification of NeuN-positive cells shows that Tau-/- makes AICD-Tg mice resistant to KA-mediated neurodegeneration. F. AICD-Tg mice show an increase in neuropeptide-Y (NPY) expression in mossy fiber terminals (arrows) following KA-mediated seizures. Tau deletion decreased NPY expression in mossy fiber terminals following KA injection in AICD-Tg animals. G. Quantification of NPY immunoreactivity normalized to WT animals. *p<0.05, **p<0.01, ***p<0.001, one way ANOVA (all data expressed as Mean ± SEM). n = 5 for all groups (D-G). Scale bar = 50 μm (D) and100 μm (F).</p