As with industrious employees, it is hard to spur greater productivity in an enzyme by simply cracking the whip. However, clearing distractions might do the trick. Taking this strategy into an Alzheimer disease mouse model, scientists have boosted the activity of an Aβ-degrading enzyme by reducing levels of an endogenous inhibitor. The enzyme, cathepsin B (CatB), caught the attention of Li Gan at the Gladstone Institute of Neurological Disease in San Francisco several years ago when it appeared to prevent buildup of amyloid plaques in the brains of AD mice overexpressing mutant human amyloid precursor protein (APP). In the October 23 Neuron, Gan and colleagues now report that APP mice lacking cystatin C (CysC)—an inhibitor of cysteine proteases including CatB—have lower soluble Aβ levels and reduced Aβ-associated deficits in cognition, behavior, and synaptic plasticity compared to APP/CysC+/+ mice. By crossing the animals onto a CatB-null background, they show that these benefits depend on CatB. In light of prior human polymorphism data and recent mouse data highlighting cystatin C’s ability to interfere with amyloid aggregation, the new findings suggest a more complex character for cystatin C and make it challenging to say whether it plays a “good” or “bad” role in AD pathogenesis.
In the same previous study that found greater amyloid deposition in CatB-deficient APP mice (see ARF related news story), Gan’s group showed that CatB trims Aβ1-42 at its C-terminus, leaving behind smaller, less toxic Aβ peptides. The scientists therefore reasoned that they could enhance CatB activity by removing an endogenous inhibitor, CysC. Indeed, when they examined hippocampal lysates from CysC+/+, +/- and -/- mice (Huh et el., 1999), first author Binggui Sun and colleagues found gene dose-dependent increases in CatB activity in the CysC-deficient animals. Next, they crossed the CysC knockouts with hAPP-J20 transgenic mice, a human mutant APP overexpressing line with increased Aβ42 production. At two to four months of age, prior to Aβ deposition, CysC-deficient APP mice had lower levels of total soluble Aβ and Aβ1-42. “That gave us confidence that we were really looking at Aβ metabolism without the effects of Aβ stabilization,” Gan told ARF. Both processes influence plaque formation, she noted, making it hard to distinguish these effects in older mice where amyloid deposition is well underway.
Consistent with their findings in young, predeposition animals, the researchers found reduced Aβ deposits in immunostained hippocampi from APP/CysC-/- mice, compared to APP/CysC+/+ animals, at five to eight and eight to 10 months of age. However, in experiments using thioflavin S, which predominantly labels Aβ fibrils and spares diffuse Aβ forms, they saw Aβ reduction in CysC-null APP mice at five to eight months but not at eight to 10 months of age. This suggested to the authors that CysC may affect soluble and fibrillar Aβ forms differently.
The Aβ trends found support in several lines of functional evidence. The researchers showed that CysC ablation in the APP mice prevented premature mortality and increased expression of calbindin (a neuronal marker whose decreased levels correlate with cognitive decline in APP-J20 mice). Furthermore, loss of CysC reduced Aβ-associated cognitive defects (revealed by Morris water maze tests) and restored long-term synaptic plasticity in hippocampal circuits involving the dentate gyrus.
To show that the benefits of CysC reduction are in fact mediated by bolstering CatB activity, the researchers crossed the APP mice (expressing +/+, +/- or -/- CysC) onto a CatB-null background. As predicted, the effects on calbindin immunostaining and hippocampal synaptic function in the CysC-deficient APP mice disappeared when CatB was out of the picture. In line with these observations, CysC deficiency had no effect on total soluble Aβ or Aβ1-42 levels in the hippocampus of APP mice lacking CatB. However, on the CatB-null background, hippocampal plaque load in four- to six-month-old APP/CysC-/- mice was about 50 percent higher than in APP/CysC+/+ animals. At first glance, this trend would seem to back recent work indicating a seemingly beneficial role for cystatin C in AD mice—as an agent that interferes with amyloid aggregation (see ARF related news story). But Gan thinks the new data are “completely consistent with the argument that cystatin C fulfills at least two functions”—inhibiting CatB and blocking aggregation by binding to Aβ. “Once you’ve taken CatB off the table, cystatin C’s other effects (i.e., its influence on Aβ aggregation) will be more prominent,” she told ARF.
More work is needed to tease out which mechanism plays a bigger role in the AD brain. Meanwhile, data on human polymorphisms thus far seem to point to a protective role for cystatin C in AD. As suggested by several studies (Benussi et al., 2003; Paraoan et al., 2004), a cystatin C variant (A25T) that is associated with higher AD risk appears to reduce production and secretion of the protein. Furthermore, in a study of 761 older men published last month in Neurology (Sundelöf et al., 2008), a 0.1-mumol/L drop in serum cystatin C between ages 70 and 77 years correlated with a 29 percent higher risk of incident AD. “In humans, the evidence is that reducing cystatin C levels puts you at risk for AD,” said Paul Mathews of the Nathan Kline Institute at New York University School of Medicine. “That would be more in line with the idea that small increases in cystatin C levels could be protective.”
Mathews noted in an ARF interview that differences in the AD mouse models used for the various cystatin C studies may be important. “If cathepsin B's activity is to chew back at the C-terminus of Aβ, particularly Aβ42, then a mouse with increased Aβ42 production (the APP-J20 mouse) will show the greatest effects when cathepsin B activity is increased,” he said. “I think that this may be a somewhat more artificial situation than that in the CNS of the APP23 and Tg2576 mice, where Aβ40:Aβ42 ratios are more like in the human.” Mathews was a coauthor on a recent study (Mi et al., 2007) led by Efrat Levy, also at Nathan Kline, which showed lower Aβ deposition in Tg2576 mice that overexpress cystatin C. Published at the same time, an independent study by Mathias Jucker’s group at the Hertie Institute for Clinical Brain Research in Tuebingen, Germany, found similar Aβ-reducing effects of cystatin C in APP23 mice (Kaeser et al., 2007).
Jucker found more inconsistencies with the apparent benefits of CysC reduction in the current study. He told ARF that his group detected no change in levels of mouse Aβ40 and Aβ42 in CysC-/- mice relative to CysC+/+ animals. Furthermore, Gan said her team found a mere 20 percent reduction (not statistically significant) in mice that overexpress human cystatin C. She believes these puzzling results could stem from the high physiological concentrations of CysC relative to CatB. The CatB-dependent effects would likely be masked in CysC-overexpressing mice because endogenous levels of the protein are already in relative excess, she said.
To see whether the current observations hold in an independent mouse model, Gan and colleagues are crossing the CysC-deficient mice with transgenics expressing wild-type APP. In the meantime, they are working to nail down where in the cell the CysC-CatB interactions take place. “It’s very surprising to us that when you take out cystatin C, a major cysteine protease inhibitor, you see no toxic effects,” Gan said. “Maybe the (CysC-CatB) interaction only happens in a compartmentalized way.” She suspects the endosome of being that place, since Aβ colocalizes with cystatin C there, and its low pH is optimal for CatB activity.
On the translational front, Gan’s group is planning a high-throughput screen for small-molecule compounds that inhibit CysC-CatB interactions—either by directly blocking the association or inducing a conformational change in one protein that affects its binding to the other. “That could be a pharmacological way to unleash the CatB-dependent Aβ degradation,” she said.—Esther Landhuis