10 September 2012. Cellular stress has been implicated in many neurodegenerative diseases, but little is known about how its pathways interact with pathology. Now, in the September 6 Neuron, researchers led by Sung Yoon and Kun Huang at The Ohio State University, Columbus, report that Aβ oligomers can activate metabolic stress pathways by blocking protein translation. The increased stress turns on a kinase involved in cell death, JNK3, which then acts to ramp up production of Aβ, the authors report. This harmful feedback loop might make JNK3 a therapeutic target, Yoon proposed. “Our data strongly suggest a positive feed-forward mechanism that is perpetuated by JNK3, so if we halt it in some manner, maybe we could significantly lower [Aβ levels],” she told Alzforum.
Cellular stressors, such as hoards of misfolded proteins, trigger the unfolded protein response (UPR), a coping mechanism that switches on early in conditions such as amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease (see ARF related news story). As part of this response, cells activate JNK (see Urano et al., 2000). Previous work by Yoon and others showed that the brain-specific isoform JNK3 mediates neuronal death in Parkinson’s disease (see Hunot et al., 2004) and Huntington’s disease (see ARF related news story), as well as the death of neurons and oligodendrocytes after brain injury (see Beffert et al., 2006; Li et al., 2007). “The new study is thorough and presents compelling evidence that Aβ can play a role in activating JNK3,” said Uwe Beffert, Boston University, Massachusetts. Beffert has collaborated with Yoon in the past but was not involved in the current research.
The route from Aβ to the kinase is an indirect one, however. Yoon and colleagues first showed that the peptides suppress the translational machinery that synthesizes proteins from messenger RNA. In rat hippocampal neuron cultures treated overnight with synthetic Aβ42 peptides (5 μM), protein synthesis dropped by about half. The authors traced the mechanism to the activation of AMP-activated protein kinase α (AMPKα), which is known to block initiation of translation (see Gwinn et al., 2008; Inoki et al., 2003). Because translation sputters, it kicks off the unfolded protein response and activation of JNK3, the authors report. How Aβ might activate AMPKα is not clear, but the authors speculate the peptide may pump up calcium levels, thereby activating CaMK, which can phosphorylate AMPKα. Intriguingly, Aβ has been shown to flood cells with calcium from internal stores, triggering the UPR (see ARF related news story).
What form of Aβ is responsible for blocking translation? Western blots of the peptide solution showed that most of it was monomeric, with about 5 percent being small oligomers, mostly dimers and trimers, the authors report. Control experiments showed that fibrillar and pure monomeric Aβ did not activate AMPKα, implying that oligomers were the active species in these experiments. Their concentration in the mixture was about 250 nM, which is higher than some estimates of physiological concentrations of Aβ42 in AD brains. The field has grappled with the issue of what is the physiologically relevant form and concentration of Aβ (see ARF Webinar).
Yoon and colleagues then turned to in-vivo studies. They crossed 5xFAD transgenic mice with JNK3 knockout mice, and saw dramatic improvements in pathology. At one year old, the 5xFAD/JNK3 knockouts accumulated about one-quarter the amount of insoluble Aβ in multiple brain regions as did their JNK3-positive cousins. Fewer cortical neurons died in the absence of JNK3 than in transgenic controls, although still more than in wild-type animals. Memory in a fear paradigm approached wild-type levels in the crosses. This suggests a role for JNK3 in promoting AD-like pathology and neuronal damage in mice.
The authors also examined where JNK3 is expressed and what it does in 5xFAD transgenics. Immunohistochemistry showed that phosphorylated JNK3 loiters near amyloid plaques and dystrophic neurites in 5x mice, Tg2576 mice, and aged monkeys. They found that at six months of age, JNK3 is about 25 percent more active in transgenics than in wild-type mice, an increase similar to what is seen in human postmortem AD brains. Both findings jibe with previous reports that JNK3 switches on in damaged neurites (see, e.g., Muresan and Muresan, 2005; Cavalli et al., 2005), and places the kinase in a relevant location to play a role in AD.
Finally, the authors linked the kinase to altered processing of amyloid precursor protein (APP). In cell culture experiments, JNK3 phosphorylated APP at position T668, as shown previously by other groups. Treating cultures with JNK activators rapidly increased APP at the cell surface, followed by a gradual reuptake that cut the amount of surface-bound APP in half within 30 minutes. This implied to the authors that JNK3 facilitates the internalization of APP. Since amyloidogenic processing is believed to occur largely in endosomal compartments, this would pump up Aβ levels, the authors note. Mutation of the T668 phosphorylation site, or treatment with a JNK inhibitor, prevented the response.
JNK3-negative 5xFAD mice displayed less phosphorylated APP at the cell membrane than normal 5x animals, but had normal APP phosphorylation in whole-cell lysates. The authors suggest that JNK3 preferentially affects membrane-bound APP. However, Beffert noted that the change in phosphorylated APP is small, and questioned whether it could lead to the large changes seen in insoluble Aβ levels. JNK3 may be acting on other substrates that affect amyloid processing as well, he suggested.
Yoon believes that JNK3 could make a therapeutic target. She plans to test inhibitors in several strains of AD mice to see if they lower Aβ levels and improve symptoms, particularly when given late in the disease course.—Madolyn Bowman Rogers.
Yoon SO, Park DJ, Ryu JC, Ozer HG, Tep C, Shin YJ, Lim TH, Pastorino L, Kunwar AJ, Walton JC, Nagahara AH, Lu KP, Nelson RJ, Tuszynski MH, Huang K. JNK3 perpetuates metabolic stress induced by Aβ peptides. Neuron. 2012 Sep 6;75(5):824-37. Abstract