We have shown here that two Aβ-reducing therapies, Aβ1–42 vaccination and NCX-2216, were equally effective in reducing Aβ deposition both in the compact and diffuse states. However, administration of the two therapies together did not increase amyloid removal. Active immunization with Aβ increased levels of CAA and microhemorrhage, an effect not observed with NCX-2216 treatment. NCX-2216 did not attenuate this potentially adverse effect of Aβ vaccination.
NCX-2216 has been shown previously to reduce amyloid deposition in APP+PS1 transgenic mice. Jantzen et al (2002)
showed that 5 months of administration of NCX-2216 in the diet of APP+PS1 transgenic mice significantly reduced amyloid load. In the current study we show that nine months of treatment with NCX-2216 significantly reduced amyloid load as detected by Aβ immunohistochemistry, in which the majority of stained deposits are diffuse, and Congo red, detecting compact amyloid deposits. These reductions were similar in the percent reductions to that observed previously (Jantzen et al, 2002
). The mechanism by which NCX-2216 reduces amyloid deposition remains unclear. It has been shown in vitro that some NSAIDs can reduce Aβ production via a γ-secretase inhibition unrelated to cyclooxygenase inhibition (Weggen et al, 2001
). It has also been shown that NCX-2216 activates peroxisome proliferator-activated receptor-γ (PPAR-γ much more effectively than the parent NSAID, flurbiprofen (Bernado et al, 2006
), suggesting another mechanism for Aβ reductions since pioglitazone, a PPAR-γ agonist, has also been shown to reduce Aβ in vivo in a transgenic mouse model (Heneka et al, 2005
), possibly by down-regulating β-secretase RNA expression (Sastre et al, 2006
active vaccination has previously been shown to produce anti-Aβ antibody titers and reduce amyloid deposition in transgenic mice (Schenk et al, 1999
). It has also been shown to attenuate memory loss in APP+PS1 transgenic mice (Morgan et al, 2000
) and in singly transgenic APP mice (Janus et al, 2000
). We have also shown that there appears to be a dependence on microglial activation for the removal of compact, Congophilic amyloid deposits following active vaccination (Wilcock et al, 2001
) and following direct intracranial injection of anti-Aβ antibodies (Wilcock et al, 2003
). In the current study we show that Aβ vaccination results in generation of significant anti-Aβ antibody titers, reduced amyloid deposition and increased microglial activation associated with the remaining compact amyloid deposits. It has been suggested that Aβ immunotherapy may either prevent further amyloid accumulation or remove existing deposits. The current study is inconclusive with regard to this issue. While the amyloid loads in the Aβ1–42
vaccinated mice are greater than we have previously observed in 10 month old mice (the age of the mice at the start of the current study) (Gordon et al, 2002
) it is unclear whether results primarily reflect a partial prevention of amyloid deposition or whether removal and turnover of some deposits is occurring.
Interestingly, we also show that there is an apparent increase in CAA and CAA-associated microhemorrhage following vaccination. We have previously shown that this phenomenon is occurring following passive immunization with Aβ antibodies (Wilcock et al, 2004c
). Other groups have also shown that treatment of aged APP transgenic mice with anti-Aβ antibodies results in an increased incidence of vascular microhemorrhage (Pfeifer et al, 2002; Racke et al, 2005
). However, to date, this vascular adverse event has not been reported following active immunization. Some studies examining anti-Aβ immunotherapy using transgenic mice have examined the brains for microhemorrhage and have not detected such events. One such study used a different transgenic mouse model (J20 APP transgenic mouse) and only examined them at 10 months of age, following 6 months of vaccination (Seabrook et al, 2006
). It would appear that age is a significant factor in the development of immunotherapy-associated microhemorrhage as Jucker and colleagues (2002) showed following passive immunotherapy in old and young APP transgenic mice. Only the old mice with significant CAA developed the microhemorrhages while 6 month old mice receiving the same passive immunization protocol failed to show any microhemorrhage. A study by Racke and colleagues (2005)
also demonstrated that the occurrence of microhemorrhage is dependent on the antibody being used. 22-month-old PDAPP transgenic mice were passively immunized for 6 weeks with either 266 (mid-domain antibody; does not bind plaques) or 3D6 (N-terminal antibody; binds plaques) anti-Aβ antibodies and found that only the 3D6 antibody resulted in an increased incidence of CAA. The authors suggest that the results indicate plaque binding is necessary for increased microhemorrhage to occur. We have previously shown that the anti-Aβ antibodies produced following the active vaccination protocol used here are primarily N-terminal antibodies with Aβ reactivity being competed by soluble Aβ1–40, Aβ1–42
and Aβ1–16 (Dickey et al, 2001
), but not by Aβ 10–20, 20–29 or 29–40. We also showed that active vaccination with this regimen produces antibodies primarily of the IgG1 and IgG2b isotypes, along with low levels of IgG2a (Dickey et al, 2001
Active Aβ vaccination advanced to phase 2 clinical trials where dosing was terminated due to 6% of the patients developing meningoencephalitis (Orgogozo et al, 2003
). Despite the trial being halted, the patients continued to be observed and some continued to produce anti-Aβ antibodies. Two autopsy reports from patients in the trial showed that those who developed meningoencephalitis had T-cell infiltration in the brain (Nicoll et al, 2003
; Ferrer et al, 2004
). Of the three autopsy reports published to date, all suggest that the severity of CAA is greater in vaccinated patients than the average for AD patients of the same stage (Nicoll et al, 2003
; Ferrer et al, 2004
; Masliah et al, 2005
). One of these reports also notes the presence of “multiple cortical hemorrhages” in the brain (Ferrer et al, 2004
) while a second indicates the CAA is "severe” (score of 3) typically indicating hemorrhage is present (Masliah et al, 2005
While one might hypothesize that a lower dose of antibody may prevent the occurrence of vascular adverse events due to immunotherapy, it appears that even relatively low levels of anti-Aβ antibodies over time are sufficient to produce CAA associated microhemorrhage. Moreover, the NCX-2216 treatment alone reduced amyloid deposits to a similar degree as the vaccine, yet did not produce the increase in vascular deposits. Given the presence of increased vascular staining with both active and passive immunization, it would appear that something associated with the antibody -mediated clearance is responsible for the increase in CAA, rather than amyloid reduction per se.
Based on approximate calculations, the titers (8 μg in 1ml serum) achieved in the current active vaccination study appears to equate to a 3mg/kg dose of anti-Aβ antibodies when translating to a passive immunization protocol. This is 30% of the dose we have previously shown (using passive immunization) to result in microhemorrhage and exacerbation of CAA following three months of anti-Aβ antibody treatment (Wilcock et al, 2004c
). Using a similar active vaccination regimen to the one reported here, we have previously shown that antibody titers reach their maximum levels following 7 immunizations. Following inoculation cessation, antibody titers declined only 20% after two months, with detectable titers still present 14 months after the last immunization (Dickey et al, 2001
). Therefore, we would not predict that the antibody titers were substantially greater earlier in the study, although it is plausible that serum antibody titers were slightly higher immediately following the final immunization. Together with our previous studies (Wilcock et al, 2004c
), the data presented here suggest that the levels of anti-Aβ antibody may not be as significant as duration of treatment and mouse age when increasing vascular amyloid with immunotherapy.
Furthermore, treatment with an NSAID along with the active Aβ vaccination did not attenuate the vascular consequences of vaccination, nor did it impair clearance. In our earlier work with intracranially applied anti-Aβ antibodies, we found that NCX-2216 partially attenuated both the activation of microglia and the clearance of compact amyloid deposits caused by the antibody (Wilcock et al, 2004a
). In contrast, dexamethasone completely suppressed both effects, implying that the NCX-2216 does not maximally suppress microglia in vivo.
Administration of NCX-2216 with Aβ1–42
vaccination did not result in greater amyloid removal than either treatment alone. It is plausible that there is a limit to the extent of amyloid removal and a dynamic equilibrium exists between Aβ removal and production. We have observed what appears to be an equilibrium effect previously following intracranial LPS administration, where amyloid was removed within seven days, but returned to control levels by four weeks. In essence, the mouse accumulated the same amount of amyloid in three weeks that originally accumulated over 16 months (Herber et al, 2004
). There are three proposed mechanisms for antibody-mediated Aβ removal. These are Fc-receptor mediated microglial phagocytosis (Bard et al, 2003
), efflux of Aβ from the brain into the plasma (DeMattos et al, 2001
), possibly via the FcRn receptor (Deane et al, 2005
) and a direct, catalytic disaggregation of amyloid deposits by the antibody (Solomon et al, 1997
). The proposed mechanisms of NSAID-mediated reductions in Aβ (vide supra) do not in any way overlap with the mechanisms of antibody-mediated reductions. It is possible that antibody-mediated microglial activation results in a phenotypic switch to an activated microglial cell state where NSAIDs no longer have an effect. We have previously suggested that microglia may exist in distinct activation states, one of which may actually be beneficial in AD (Morgan et al, 2005
In summary, we have shown here that two amyloid-lowering therapies administered together do not show additive or synergistic effects in their amyloid-lowering abilities. However, importantly, we show that active immunization with Aβ1–42
increases levels of CAA and microhemorrhage. This effect is not attenuated by an NSAID such as NCX-2216. While it is unclear what impact microhemorrhages have clinically in AD, it has been shown that CAA is associated with intracerebral hemorrhages as well as ischemic infarcts (Attems, 2005
). While we have previously shown that microhemorrhage as a result of immunotherapy does not impact mice cognitively (Wilcock et al, 2004c
) the data presented here should increase concern with respect to ongoing clinical trials that even low levels of circulating anti-Aβ antibodies may result in vascular complications with long durations of treatment.