A cat might be the last thing a sickly AD mouse needs, particularly if it is CatB (cathepsin B), a papain-family lysosomal cysteine protease suspected of contributing to the production of amyloid-β (Aβ). But in an ironic twist, a new paper reveals an entirely different CatB/mouse relationship. Li Gan and colleagues at the Gladstone Institute of Neurological Disease and the University of California at San Francisco report that CatB not only degrades soluble Aβ, but attacks fibrillar forms as well, a rare talent among the proteases that might clear Aβ in vivo. The work appears in this week’s Neuron.
The original idea that cathepsin B (CatB) might be a secretase stems from its location in endosomes and lysosomes where Aβ is produced. In addition, CatB has been found in enlarged endosomes and neuritic amyloid plaques in AD brain. To test the secretase hypothesis, first author Sarah Mueller-Steiner and colleagues crossed their human APP mouse with CatB knockouts (see ARF related news story). If CatB were a secretase, they reasoned, the hAPP/CatB-/- mice should have fewer plaques than their cathepsin-replete littermates. Instead, the researchers found the precise opposite—young mice lacking CatB displayed more, not fewer, amyloid plaques in their hippocampus and cortex. The increased plaque load was accompanied by a small increase in total Aβ and Aβ1-42 levels which did not reach statistical significance. However, the relative ratio of Aβ1-42 compared to total Aβ was significantly elevated. The mice also had increased neuropathology, as indicated by decreased staining for the synaptic calcium binding protein calbindin: the same group previously showed that calbindin loss correlates tightly with cognitive decline in this strain of mice (see ARF related news story). When the authors suppressed CatB in cultured primary mouse neurons, they found that levels of both Aβ1-42 and total Aβ increased, while boosting the protease had the opposite effect. No effect was seen on APP processing, suggesting that CatB influenced Aβ degradation.
Cathepsin B has been reported to localize to plaques (reviewed in Nixon and Cataldo, 2006), so the researchers looked for the enzyme in the brains of hAPP mice. They found that in older hAPP mice, cathepsin was associated with 20 percent of all plaques and 70 percent of mature neuritic plaques, which contained fibrillar Aβ, as indicated by thioflavin S staining. In younger mice, most plaques, both diffuse and neuritic, were cathepsin-positive. They also found the enzyme in both neurons and activated glia surrounding the plaques, and that all three cell types—neurons, astrocytes and microglial cells—secreted the enzyme in culture. In neurons, CatB localized to the lysosomal/endosomal compartment.
Could induction of CatB represent a protective mechanism against Aβ toxicity? Aβ1-42 induces CatB mRNA in microglia (Gan et al., 2004), and when the authors added Aβ1-42 to N2A neuronal cells in culture, they also observed an increase in CatB mRNA and activity. Aβ1-40, however, had no effect on neuronal CatB unless it was pre-aggregated. Consistent with these in vitro results, the investigators observed higher CatB activity in the hippocampus of young and middle-aged hAPP mice. This induction might fail with aging, as they did not see upregulation of CatB in older hAPP mice. Their results are consistent with the idea that defects in Aβ clearance with aging might underlie amyloid accumulation.
In vitro studies showed that CatB clips Aβ1-42 at its C-terminus, leaving the less amyloidogenic, and therefore less toxic, Aβ1-40, Aβ1-38, and Aβ1-33 peptides as products. The enzyme appeared to cleave Aβ1-42 sequentially, first to Aβ1-40, then to Aβ1-38. Higher concentrations of enzyme yielded Aβ1-33, but did not completely degrade the peptide. Interestingly, CatB also cleaved aggregated Aβ into less fibrillogenic species, eliminating the high molecular mass assemblies that are detected on polyacrylamide gels. Cleavage by CatB was higher at pH 6, the conditions inside lysosomes where it might encounter Aβ. But the enzyme also degraded Aβ at pH 7, suggesting it could work outside cells as well.
To ask the all-important question of whether CatB can reduce established plaques in hAPP mice, the researchers used lentiviral vectors to express the enzyme in the brains of 12-15-month-old hAPP mice. After 3 weeks, they detected a boost in CatB activity, and reduction in plaques. CatB was as effective at reducing plaques as a neprilysin construct introduced in the same way, and was more effective than neprilysin at removing thioflavin S-positive plaques, suggesting its superior ability to remove aggregated Aβ. Recently, matrix metalloprotease 9 was also shown to degrade Aβ fibrils in vitro (Yan et al., 2006); this capacity is not shared by Aβ degraders—insulin-degrading enzyme, neprilysin, and plasmin.
In an accompanying commentary, Greg Cole and Sally Frautschy of the University of California at Los Angeles write that the identification of a major protease responsible for intracellular and extracellular clearance of Aβ assemblies is full of promise, and they discuss the possibility of using this information to therapeutic advantage. “For example, the authors observed that microglia secrete abundant CatB. This may be regulated by their activation state, which, for example, can be upregulated by the amyloid vaccine or anti-amyloid antibodies, currently one of the most effective methods of reducing preformed thioflavin S-labeled amyloid plaques,” they write. But they also caution that CatB might contribute to neurotoxic effects of microglia. The first step must be finding out if CatB plays a similar role in humans as it does in the mouse.—Pat McCaffrey
- The Limits of Compensation: Double Loss of Proteases Causes Massive Neuronal Death
- Calbindin Study: Is Calcium the Molecular Handle on Dysfunction in AD?
- Nixon RA, Cataldo AM. Lysosomal system pathways: genes to neurodegeneration in Alzheimer's disease. J Alzheimers Dis. 2006;9(3 Suppl):277-89. PubMed.
- Gan L, Ye S, Chu A, Anton K, Yi S, Vincent VA, von Schack D, Chin D, Murray J, Lohr S, Patthy L, Gonzalez-Zulueta M, Nikolich K, Urfer R. Identification of cathepsin B as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach. J Biol Chem. 2004 Feb 13;279(7):5565-72. PubMed.
- Yan P, Hu X, Song H, Yin K, Bateman RJ, Cirrito JR, Xiao Q, Hsu FF, Turk JW, Xu J, Hsu CY, Holtzman DM, Lee JM. Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ. J Biol Chem. 2006 Aug 25;281(34):24566-74. PubMed.
- Mueller-Steiner S, Zhou Y, Arai H, Roberson ED, Sun B, Chen J, Wang X, Yu G, Esposito L, Mucke L, Gan L. Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.
- Cole GM, Frautschy SA. Cat and mouse. Neuron. 2006 Sep 21;51(6):671-2. PubMed.