These forms of Aβ are not only more stable than the unmodified peptide, but also more fibrillogenic, making them potentially more dangerous to have lurking in one’s brain. This was a point Demuth emphasized. He showed that while pGluAβ3-40 undergoes fibrillization faster than Aβ40, even further acceleration occurs when pGluAβ3-40 is used to seed solutions of Aβ40. This hints that in the AD brain, even a small amount of pGluAβ3-40, or of the even more amyloidogenic pGluAβ3-42, may be sufficient for such a snowball effect. A similar dynamic was seen pGluAβ3-38 in comparison to Aβ38, raising the question of whether γ-secretase modulation to increase Aβ38 production at the expense of Aβ42 production will by itself have the desired effect on amyloid pathology.
To test the relevance of this idea, Demuth and colleagues, together with Thomas Bayer and colleagues at the University of Göttingen, Germany, have created a transgenic mouse model where the third position of Aβ, the glutamate (E), is changed to a glutamine (Q). Glutamine gets cyclized more than three orders of magnitude faster by endogenous glutaminyl cyclase than is glutamate, so if N-terminal pyroglutamate is important for neurodegeneration, one might expect these animals to have accelerated pathology. This is exactly what Demuth showed at Keystone. These transgenic animals, called TBA2, show extremely rapid onset of symptoms, having behavioral problems and motor activity deficits (they cannot find or reach food) by the age of two months. At the same age, the animals’ hippocampus and cortex reacts with antiserum that recognizes both monomeric and oligomeric Aβ.
The Aβ in these animals is produced as part of a prothyrotropin-releasing hormone (proTRH)-Aβ chimera driven by a brain-specific promoter. The N3QAβ is cleaved from proTRH-Aβ by prohormone convertases in the secretory pathway. A similar construct (release of Aβ by furin from a BRI-Aβ sequence) was used by Eileen McGowan and colleagues at the Mayo Clinic, Jacksonville, Florida, to circumvent the complications of γ-secretase cleavage at both the 40 and 42 positions. McGowan and colleagues found that Aβ40 produced from this construct is actually neuroprotective (see ARF related news story). That N3QAβ driven from a similar system is destructive is indicative of the toxicity of pGluAβs. “This is the first Alzheimer’s-related model that shows a tremendous behavioral phenotype after only two months of existence,” said Demuth. He further demonstrated the toxicity of N3QAβs in hippocampal rat brain slices. Conditioned medium from HEK293 cells transfected with APP-NLQ (a construct having asparagine, leucine, and glutamine in the first three positions of Aβ) more strongly depressed hippocampal LTP than did medium from cells expressing normal APP or APP-NLE, which would yield full-length Aβ or Glu3Aβ with slower N-terminal cyclization rates. To Demuth, the in-vivo and tissue culture experiments together confirm the toxicity of pGluAβ.
Human Glutaminyl Cyclase—A Therapeutic Target?
If pGluAβ drives AD pathology, then what is driving cyclization of the N-terminal glutamate in the diseased state that is not at work in normal brain? Possible answers here include enhanced Aβ production, enhanced removal of the N-terminal dipeptide, or more rapid cyclization.
Glutaminyl cyclase (QC), the enzyme that cyclizes N3Aβ glutamine in the above mouse model, is normally found in the brain, but at Keystone, Demuth presented data to suggest that this enzyme is massively overexpressed in AD brain. Enzyme and pGluAβ levels show the same pattern, with QC levels being increased up to 40 times depending on the severity of the investigated stages. The enzyme is expressed in brain areas affected in AD, including the CA3 layer of the hippocampus and the cortex.
Such data hint that targeting QC might be one approach to tackling AD pathology, and that’s just what Probiodrug has in mind. Demuth presented data on one experimental inhibitor, PBD150, suggesting efficacy in animal models. Used as a prophylactic in Tg2576 mice beginning at four or six months, PBD150 reduced both pGluAβ and total Aβ, and it improved memory in a contextual fear paradigm. Its efficacy was similar when used as a treatment in older (10 months) animals that have formed plaques. PBD150 reduced pGluAβ and improved memory. Prophylaxis worked in the TASD41 mouse, which produces very high amounts of human APP carrying both Swedish and London mutations (see Rockenstein et al., 2001). Demuth said that Probiodrug has profiled better compounds for passage of the blood-brain barrier.
One potential bonus of QC inhibition is that it may also reduce reactive gliosis. In characterizing the effect of PBD150, Demuth’s collaborators Steffen Rossner at the Brain Research Institute at University of Leipzig, and Manfred Windisch of the contract research organization JSW Research in Graz, Austria, noticed reduction in the number of reactive glia surrounding plaques in treated Tg2576 and TASD41 mice, respectively. This may simply be due to the reduction of plaque load or the reduction of seed peptides, but it may also be related to a direct inhibition of glial responses. In this regard, Demuth noted that other pyroglutamate peptides, in particular pGlu derivatives of monocyte chemoattractant protein (MCP-1), which has an N-terminal glutamine, trigger glial cell migration. Therefore, blocking QC may also reduce inflammatory responses in the brain and even elsewhere. Probiodrug is screening QC inhibitors for inflammatory diseases such as atherosclerosis, as well.
Could this anti-inflammatory aspect be a downside to the strategy, leading to side effects? “The binding constants for the cyclization of glutamine peptides are much lower than for glutamate peptides, so you don’t have to fully inhibit QC’s negative side function of glutamate cyclization within a certain time frame to get an effect on Aβ but still have sufficient glutamine cyclization for maturation of proteins, for example, peptide hormones,” said Demuth.
There may be other surprises in store from this pyroglutamate story. Demuth briefly described experiments that lead him to believe BACE may not be the only important enzyme that leads to Aβ production. This possibility has been entertained before but currently represents a fringe view (see ARF related news story). Cultured HEK293 cells produce much less pGluAβ when transfected with APP containing the Swedish mutation, which promotes cleavage at the BACE site. This suggests that there may be another protease that cleaves Aβ in such a way that the glutamate is unmasked at the N-terminus and can be cyclized. One possibility is that an alternate protease cleaves APP at the three position of Aβ.
Whether another protease is important in vivo remains to be seen. Robert Vassar of Northwestern University, Chicago, attended the meeting but is not involved in this work. He told ARF that the PDAPP mouse used by Elan is wild-type at the β-secretase cleavage site and, when crossed with BACE knockouts, produces no Aβ or amyloid plaques, suggesting that BACE is the major protease for Aβ even in wild-type APP (McConlogue et al., 2007). “There are other enzymes that cut [at the β site], but they cut rarely and the amounts of their Aβ are very low, so I don’t think they play a significant role in AD. They do start to cut at higher levels when you overexpress APP. It is possible that under condition of overexpression, and when you inhibit BACE so you have more full-length APP, that some of these aberrant cleavages appear to increase,” Vassar suggested. Perhaps any BACE doppelganger will reveal itself at the next Keystone meeting.—Tom Fagan.