Activation of JNK Signaling Mediates Amyloid-ß-Dependent Cell Death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone.Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies

Misexpression of Aß42 triggers cell death in the differentiating neurons.

<p>(A, A′) Wild-type third instar larval eye imaginal disc displaying randomly distributed TUNEL positive dying cells (A, A″) shown in red channel (arrow). TUNEL staining marks the fragmented DNA within the nuclei of dying cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Singh1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-White1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-McCall1" target="_blank">[61]</a>. (B) Wild-type adult eye. (C, C′) Misexpression of Aß42 (GMR>Aß42) in differentiating neurons of the eye show elevated levels of TUNEL positive cells (C′ arrows). The increased frequency of cell death in neurons can be directly correlated to the misexpression of the Aß42 peptide. Note that misexpression of Aß42 does not affect the differentiation process as the distribution of Elav positive cells is the same in both control and Aß42 third instar eye imaginal discs. (D) GMR>Aß42 results in a strong neurodegenerative phenotype in adult eye. Baculovirus P35 has been shown to block the caspase dependent cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Hay1" target="_blank">[36]</a>. (E, E′, F) Misexpression of P35 along with Aß42 in differentiating neurons (GMR>Aß42+P35) shows significant reduction of dying cells based on number of TUNEL positive cells (red channel) in the larval eye field. However, this rescue is not as strong in (F) adult eye phenotype. (E′) Note that the eye field displays reduced number of TUNEL positive cells (arrow) compared to GMR>Aß42 eye field (C′). It is important to note that Aß42 peptide production is not affected. Elav marks the photoreceptor fate (C′″). Puckered (Puc), a dual phosphatase, is downstream target of JNK signaling pathway and forms a feedback loop to negatively regulate the pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Stronach1" target="_blank">[20]</a>. (G, G′, H) Misexpression of <i>puc</i> along with Aß42 in the differentiating neurons (GMR>Aß42+<i>puc</i>) results in significant suppression of cell death as evident from reduced number of TUNEL positive cells in the third instar larval eye imaginal disc as well as in the (H) adult eye. Note that there is a significant rescue of (D) GMR>Aß42 adult eye phenotype in (H) GMR>Aß42+<i>puc</i> background. These results suggest that JNK signaling might be responsible for neurodegeneration seen in amyloid plaque mediated cell death. (I) Quantification of the number of dying cells in eye imaginal discs based on TUNEL staining in wild-type (served as control), GMR>Aß42, GMR>Aß42+P35 and GMR>Aß42+<i>puc</i>. Note that blocking JNK signaling (GMR>Aß42+<i>puc</i>) exhibit strong rescue of the neurodegenerative phenotype of GMR>Aß42 and GMR>Aß42+P35. This rescue is significant (**) as seen by calculation of P-values based on one-tailed <i>t</i>-test using Microsoft Excel 2007.</p

Ectopic upregulation of JNK signaling induces cell death in the GMR>Aß42 eye imaginal disc.

<p>(A) Wild-type eye imaginal disc showing cell death in random cells, which serve as controls. (B) GMR>Aß42 eye imaginal disc showing ectopic upregulation of <i>puc</i> lacZ in a large number of dying retinal cells as evident from TUNEL positive staining. In comparison to the (C) wild-type adult eye, (D) GMR>Aß42 adult eye are highly reduced due to neurodegeneration. (E–L) Increasing level of JNK signaling in GMR>Aß42 by misexpressing (F, H) activated <i>hemipterous</i> (GMR>Aß42+<i>hep^Act</i>) and (J, L) activated <i>Djun</i> (GMR>Aß42+<i>jun^aspv7</i>) results in (F, J) dramatic increase in dying cell population in the eye imaginal disc, leading to a (H, L) “no-eye” phenotype in the adult fly. Increased levels of (E, G) activated <i>hemipterous</i> (GMR><i>hep^Act</i>), (I, K) activated <i>Djun</i> (GMR><i>jun^aspv7</i>) served as controls and result in some dying cells in the (E, I) eye imaginal disc and (G, K) a small adult eye. However, reducing level of JNK signaling in GMR>Aß42 background by misexpressing (N, P) Dominant negative <i>basket</i> (GMR>Aß42+<i>bsk^DN</i>) results in significant reduction to near complete absence of dying cell population (N) in the eye imaginal disc, leading to a (P) strong rescue of the adult eye phenotype as compared to GMR>Aß42 adult eye. (M, O) Increased levels of Dominant negative <i>basket</i> (GMR><i>bsk^DN</i>) in (M) eye imaginal disc and (O) adult eye served as controls. Note that increased levels of dominant negative <i>basket</i> alone (GMR><i>bsk^DN</i>) does not affect the size of eye imaginal disc and the adult eye.</p

JNK signaling is activated upon misexpression of Aß42 in the eye.

<p>(A) Schematic representation of hierarchy of Jun-kinase signaling pathway members. (B, B′) Wild-type expression of <i>puc</i> in the developing third instar larval eye imaginal disc using lacZ reporter where reporter (red channel) is restricted only to the developing photoreceptors in the eye imaginal disc proper and in the peripodial membrane cells on the margin of the antennal disc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-MartinBlanco1" target="_blank">[43]</a>. (C, C′) GMR>Aß42 eye imaginal disc exhibits ectopic upregulation of <i>puc</i>-lacZ reporter. (C′) Split channel showing ectopic <i>puc</i>-lacZ expression in the photoreceptor neurons of the eye imaginal disc. (D) Activation of JNK signaling in GMR>Aß42 was detected by checking phospho-Jun levels. Levels of JNK signaling pathway increases three fold in GMR>Aß42 as compared to the wild-type eye imaginal disc.</p

JNK signaling is responsible for cell death in GMR>Aß42 background.

<p>(A, A′) Misexpression of both P35 and <i>puc</i> along with Aß42 (GMR>Aß42+P35<i>+puc</i>) results in strong rescue of cell death as evident from (A′) dramatically reduced TUNEL positive cells. However, the rescue of the phenotype was not significantly stronger than with blocking JNK signaling pathway alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone-0024361-g005" target="_blank">Figure 5G′</a>). (B) Misexpression of Aß42 (GMR>Aß42) in pupal retina showing cell death as evident from TUNEL positive cells (red channel). Blocking simultaneously both caspase-dependent cell death and caspase-independent JNK signaling mediated cell death in pupal retina (GMR>Aß42+P35<i>+puc</i>) showed a strong rescue in (C, C′, C″) pupal retina and (D) adult eye as compared to (B) GMR>Aß42 pupal retina, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone-0024361-g003" target="_blank">Figure 3D</a>) GMR>Aß42 adult eye. The cell death is detected by TUNEL staining (red channel), which is (C′, C″) restricted to the periphery of the pupal retina. Note that dying cells on the periphery of the pupal retina corresponds to the programmed cell death as seen in the wild-type pupal retina too <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Brachmann1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Lin1" target="_blank">[28]</a>. (E) Quantification of the number of dying cells in eye imaginal discs based on TUNEL staining in different genetic combinations. The frequency of cell death in wild-type eye imaginal disc served as a control. Note that blocking JNK signaling (GMR>Aß42+<i>puc</i>) or blocking JNK signaling along with caspase–dependent cell death (GMR>Aß42+P35+<i>puc</i>) exhibit strong rescue of the neurodegenerative phenotype of GMR>Aß42. This rescue is significant (**) as seen by calculation of P-values based on one-tailed <i>t</i>-test using Microsoft Excel 2007.</p

Aß42 accumulation in the developing pupal retina and adult eye results in neurodegeneration.

<p>(A, C) represent normal eye development in (A) late pupal retina, and (C) the adult compound eye. Dlg (red channel) marks the membrane; 6E10 (blue channel) marks the Aß42, and Elav (green channel), a proneural marker that marks the neuronal fate. (B, D, F) Misexpression of Aß42 in the differentiating neurons of eye using a GMR-Gal4 construct (GMR>Aß42) results in onset of neurodegeneration. (C, C′) Unlike the highly organized ommatidia of wild-type pupal retina, (D, D′) the late pupal retina shows significant neuronal cell death in the eye field. By this stage, the accumulation of Aß42 plaques has resulted in distinct holes in the eye field (marked by an arrow, outline of “hole” in pupal retina marked by red dotted line). (C) Wild type adult eye with uniform arrangement of 800 unit eyes. (I) The adult eye field of GMR>Aß42 is significantly diminished and there is complete fusion of the ommatidia. (G, H) In comparison to the adult eye section of (G) wild type fly eye, (H) the GMR>Aß42 exhibits highly disorganized morphology of photoreceptors. Furthermore the retinas of GMR>Aß42 are vacuolated. This illustrates how the neurodegenerative phenotype progressively worsens as the age and dose increase over the course of fly development.</p