Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain

Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by γ-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3β (GSK-3β) and phosphorylation of CRMP2 protein, a GSK-3β substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding

Amyloid-Independent Mechanisms in Alzheimer's Disease Pathogenesis

Despite the progress of the past two decades, the cause of Alzheimer's disease (AD) and effective treatments against it remain elusive. The hypothesis that amyloid-β (Aβ) peptides are the primary causative agents of AD retains significant support among researchers. Nonetheless, a growing body of evidence shows that Aβ peptides are unlikely to be the sole factor in AD etiology. Evidence that Aβ/amyloid-independent factors, including the actions of AD-related genes, also contribute significantly to AD pathogenesis was presented in a symposium at the 2010 Annual Meeting of the Society for Neuroscience. Here we summarize the studies showing how amyloid-independent mechanisms cause defective endo-lysosomal trafficking, altered intracellular signaling cascades, or impaired neurotransmitter release and contribute to synaptic dysfunction and/or neurodegeneration, leading to dementia in AD. A view of AD pathogenesis that encompasses both the amyloid-dependent and -independent mechanisms will help fill the gaps in our knowledge and reconcile the findings that cannot be explained solely by the amyloid hypothesis.National Institutes of Health (U.S.) (Grant P01-AG027916)National Institutes of Health (U.S.) (Grant R01-NS051874

Knockdown of Amyloid Precursor Protein in Zebrafish Causes Defects in Motor Axon Outgrowth

Amyloid precursor protein (APP) plays a pivotal role in Alzheimer’s disease (AD) pathogenesis, but its normal physiological functions are less clear. Combined deletion of the APP and APP-like protein 2 (APLP2) genes in mice results in post-natal lethality, suggesting that APP performs an essential, if redundant, function during embryogenesis. We previously showed that injection of antisense morpholino to reduce APP levels in zebrafish embryos caused convergent-extension defects. Here we report that a reduction in APP levels causes defective axonal outgrowth of facial branchiomotor and spinal motor neurons, which involves disorganized axonal cytoskeletal elements. The defective outgrowth is caused in a cell-autonomous manner and both extracellular and intracellular domains of human APP are required to rescue the defective phenotype. Interestingly, wild-type human APP rescues the defective phenotype but APPswe mutation, which causes familial AD, does not. Our results show that the zebrafish model provides a powerful system to delineate APP functions in vivo and to study the biological effects of APP mutations

APP Intracellular Domain Impairs Adult Neurogenesis in Transgenic Mice by Inducing Neuroinflammation

A devastating aspect of Alzheimer's disease (AD) is the progressive deterioration of memory due to neuronal loss. Amyloid precursor protein (APP) occupies a central position in AD and APP-derived amyloid-β (Aβ) peptides are thought to play a pivotal role in disease pathogenesis. Nonetheless, it is becoming clear that AD etiology is highly complex and that factors other than Aβ also contribute to AD pathogenesis. APP intracellular domain (AICD) is generated together with Aβ and we recently showed that AICD transgenic mice recapitulate pathological features of AD such as tau hyperphosphorylation, memory deficits and neurodegeneration without increasing the Aβ levels. Since impaired adult neurogenesis is shown to augment memory deficits in AD mouse models, here we examined the status of adult neurogenesis in AICD transgenic mice.We previously generated transgenic mice co-expressing 59-residue long AICD fragment and its binding partner Fe65. Hippocampal progenitor cell proliferation was determined by BrdU incorporation at 1.5, 3 and 12 months of age. Only male transgenic and their respective wilt type littermate control mice were used. We find age-dependent decrease in BrdU incorporation and doublecortin-positive cells in the dentate gyrus of AICD transgenic mice suggesting impaired adult neurogenesis. This deficit resulted from decreased proliferation and survival, whereas neuronal differentiation remained unaffected. Importantly, this impairment was independent of Aβ since APP-KO mice expressing AICD also exhibit reduced neurogenesis. The defects in adult neurogenesis are prevented by long-term treatment with the non-steroidal anti-inflammatory agents ibuprofen or naproxen suggesting that neuroinflammation is critically involved in impaired adult neurogenesis in AICD transgenic mice.Since adult neurogenesis is crucial for spatial memory, which is particularly vulnerable in AD, these findings suggest that AICD can exacerbate memory defects in AD by impairing adult neurogenesis. Our findings further establish that AICD, in addition to Aβ, contributes to AD pathology and that neuroinflammation plays a much broader role in AD pathogenesis than previously thought

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

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