22 Aug 2013

Dysregulated signaling through the cellular prion protein may exacerbate pathology in both Alzheimer’s and prion diseases, according to a report in the August 18 Nature Medicine. Researchers led by Benoit Schneider at INSERM, Paris, France, found that in both disorders, a kinase downstream of the prion protein becomes overactivated. Inhibiting the enzyme, 3-phosphoinositide-dependent protein kinase-1 (PDK1, also called PDPK1), improved survival and motor skills in mouse models of prion disease, and sharpened memory in AD mice, the authors report. They traced the pathogenic mechanism to a harmful feedback loop created when PDK1 suppresses the activity of ADAM-17, an α-secretase. Normally, ADAM-17 snips amyloid precursor protein to prevent the formation of toxic Aβ fragments, and cuts prion protein so it can no longer misfold into an infectious agent. When ADAM-17 activity drops, pathogenic proteins accumulate, further activating PDK1. The data imply that the kinase could be a promising therapeutic target in AD and prion diseases if safe inhibitors can be identified, Schneider told Alzforum.

“The findings nicely fuse both diseases,” said Markus Glatzel at University Medical Center Hamburg-Eppendorf, Germany. He was not involved in the work. He noted that the data provide a pathway to explore for AD and prion drug candidates that might prove safer than inhibiting PDK1 itself. The kinase forms part of the ubiquitous Src kinase-phosphatidylinositol 3-kinase (PI3K) cascade, and has many jobs in many cell types.

Previous research suggested that cellular prion protein (PrPc), which is the normal, non-infectious form, plays a role in AD. Aβ binds PrPc with high affinity, and researchers showed this interaction mediates Aβ’s synaptic toxicity in cultures and mouse models (see ARF related news story; ARF related news story). In addition, injecting misfolded prion proteins into an AD mouse model accelerates amyloid pathology (see ARF related news story). Intriguingly, some people develop a form of AD that progresses rapidly and can be mistaken for prion disease, although it is unclear what underlying pathology characterizes this condition (see ARF related news story; ARF related news story).

Schneider and colleagues wondered if ADAM-17, also called tumor necrosis factor-α (TNF-α)-converting enzyme (TACE), might be a common link between the diseases. Reports suggest the enzyme processes both PrPc (see Vincent et al., 2001) and amyloid precursor protein (APP) (see Buxbaum et al., 1998). Once cleaved at an α site, these proteins no longer form toxic configurations. Therefore, TACE activity might help protect against disease, the authors reasoned.

They focused first on prion disease. First author Mathéa Pietri found that neurons infected with various misfolded prion strains (PrPSc) had little TACE activity compared to untreated cells, because the secretase abandoned the cell surface, where it would cleave PrP and/or APP. Pietri and colleagues traced the cause of the missing TACE to PDK1. The kinase phosphorylates the cytoplasmic tail of the secretase, leading to its internalization (for a review see Göoz, 2010). PDK1 was two to four times more active in PrPSc infected cells than control cells, the authors found. Inhibiting the kinase with the small molecule BX912 lowered TACE phosphorylation and ushered the secretase back to the cell surface. The treatment also squelched the amount of misfolded prion protein in cell cultures, presumably through increased TACE α-cleavage. Inhibiting TACE brought misfolded prion levels back up, strengthening this idea.

The authors then looked in vivo. They infected mice with scrapie prions, waited four months, and then chronically administered BX912 through an intraperitoneal osmotic pump. Treated mice lived for two months, a month more than untreated controls. Treated animals also had better motor coordination, fewer scrapie deposits, and more surviving neurons than controls. Similarly, silencing PDK1 by injecting short interfering RNA (siRNA) for seven days also reduced scrapie deposits and improved motor skills. Notably, BX912 by itself has toxic effects, killing wild-type mice within three or four months after treatment begins. Schneider speculated that these experiments may underestimate the amount of survival that could be gained by inhibiting this pathway.

Turning to Alzheimer’s, the authors examined nine-month-old Tg2576 mice. At this age, some of the animals have started to deposit Aβ in the brain. Cultured hippocampal neurons taken from animals with plaques had 150 percent more PDK1 activity than those from plaque-free transgenic mice. In these neurons TACE phosphorylation was up two-fold, and the kinase vacated the cell surface, just as in prion-infected neurons. In addition, the neurons produced twice as much Aβ40 and Aβ42 as those from plaque-free mice. All these effects could be reversed by blocking PDK1, the authors found. Treating cells with BX912 brought TACE back to the cell surface and cut Aβ levels. If they also added a TACE inhibitor, however, Aβ levels stayed high, showing that TACE activity was required to suppress pathogenic cleavage.

When Pietri and colleagues gave seven-month-old Tg2576 mice BX912 infusions for two months, fewer animals developed plaques, and those that did had fewer and smaller plaques, compared to untreated littermates. Treated animals had less Aβ40 and Aβ42 and more sAPPα in cerebrospinal fluid, consistent with increased α-cleavage. These improvements persisted for at least two months after treatment ended, the authors report. Animals that received BX912 demonstrated improved memory in a contextual fear conditioning task and the Morris water maze, and were better able to build nests than untreated animals. The authors saw the same cognitive benefits when they administered BX912 to two other transgenic AD models, 3xTg and 5xFAD mice.

What might be happening upstream of PDK1? Schneider believes that overactivation of PrPc kicks off the pathway. In prion disease, misfolded prions stimulate toxic signaling through PrPc (see, e.g., ARF related news story), and in AD, Aβ has been shown to bind and activate cellular prion protein. Moreover, PrPc is known to trigger the Src-PI3K-PDK1 pathway. To test this hypothesis, the authors silenced PrPc with siRNA in cultured AD neurons. As predicted, PDK1 activity dropped back to normal and Aβ levels fell, demonstrating that PrPc is crucial for PDK1 activation in AD. In ongoing work, Schneider is looking more closely at how PrPc signaling turns on PDK1 in these diseases. He is also examining at the structure of the TACE tail to determine exactly which phosphorylation sites control trafficking.

Finally, the authors note that the mechanism may apply in human disease because PDK1 activity is up three-fold in AD brains compared to age-matched control, and TACE activity is down 90 percent. Schneider pointed out that there are reports in the literature of amyloid deposits in the brains of people who died from prion disease, supporting the idea that overactive prion signaling increases pathogenic processing of APP (see, e.g., Barcikowska et al., 1995; Hainfellner et al., 1998; Ghoshal et al., 2009).

Other researchers said that the findings could open up new avenues for research. “The authors offer some new perspectives and new ideas,” Paul Saftig at Christian-Albrechts-University in Kiel, Germany, told Alzforum. “I think people in the prion field especially will take up these findings and try to reproduce these data.” Saftig suggested it would be informative to look at additional genetic mouse models, such as TACE knockouts or over-expressors, as well as look at other secretases. He noted that ADAM-10, not TACE, is the main α-secretase for APP. Schneider said he has some preliminary data suggesting that ADAM-10 is also dysregulated in AD neurons and prion-infected cells. Likewise, there is some controversy over which α-secretase cleaves prion protein, Glatzel noted. PDK1 might also act through another mechanism to exacerbate disease, he speculated.

Despite these questions, Schneider believes targeting PDK1 could be an effective strategy for both AD and prion diseases. Currently he is testing commercially available inhibitors for safety, and plans to design new compounds in collaboration with Hoffman-La Roche in Basel, Switzerland. One advantage of PDK1 inhibition is that it might treat advanced disease, Schneider told Alzforum. In mice, PDK1 activity goes up late in the course of both illnesses. Mice improved when treated four months after infection with prions, much later than most experimental treatments that have shown success in mouse models, he said. Schneider expects that treatment could be given in late stages of AD as well. This would distinguish this strategy from many others now under investigation, as the current trend in the field is to treat as early as possible.—Madolyn Bowman Rogers

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References

News Citations

1. Patient-Derived Aβ Needs Prion Protein to Harm Synapses 23 May 2011
2. Tracing Aβ’s Toxicity Through Prion Protein, Fyn Kinase 27 Jul 2012
3. Amyloid and Prions—Co-Conspirators in Disease? 5 Apr 2010
4. Research Brief: Does Alzheimer’s Progress Fast in Some People? 16 Sep 2011
5. Mistaken Identity—Prion Disease or Alzheimer’s on Fast Forward? 8 Feb 2013
6. Prion Protein Wields N-Terminal Flexible Tail to Kill Neurons 29 Jul 2013

Paper Citations

1. Vincent B, Paitel E, Saftig P, Frobert Y, Hartmann D, De Strooper B, Grassi J, Lopez-Perez E, Checler F. The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein. J Biol Chem. 2001 Oct 12;276(41):37743-6. PubMed.
2. Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, Black RA. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem. 1998 Oct 23;273(43):27765-7. PubMed.
3. Gooz M. ADAM-17: the enzyme that does it all. Crit Rev Biochem Mol Biol. 2010 Apr;45(2):146-69. PubMed.
4. Barcikowska M, Kwiecinski H, Liberski PP, Kowalski J, Brown P, Gajdusek DC. Creutzfeldt-Jakob disease with Alzheimer-type A beta-reactive amyloid plaques. Histopathology. 1995 May;26(5):445-50. PubMed.
5. Hainfellner JA, Wanschitz J, Jellinger K, Liberski PP, Gullotta F, Budka H. Coexistence of Alzheimer-type neuropathology in Creutzfeldt-Jakob disease. Acta Neuropathol. 1998 Aug;96(2):116-22. PubMed.
6. Ghoshal N, Cali I, Perrin RJ, Josephson SA, Sun N, Gambetti P, Morris JC. Codistribution of amyloid beta plaques and spongiform degeneration in familial Creutzfeldt-Jakob disease with the E200K-129M haplotype. Arch Neurol. 2009 Oct;66(10):1240-6. PubMed.

Other Citations

1. Tg2576

External Citations

1. PDPK1
2. 5xFAD

Further Reading

News

1. Shape-shifting Prions: Infectious Recombinant and Myelin-Minding Normal 3 Feb 2010
2. In Prion Disease, Toxicity Comes After Infection 1 Mar 2011
3. Prion Powers Synapses by Forming Amyloid 18 Feb 2010
4. Prions Abound in Yeast 10 Apr 2009
5. Prion Protein Wields N-Terminal Flexible Tail to Kill Neurons 29 Jul 2013