Robert W. Price

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

Aβ40 Inhibits Amyloid Deposition In Vivo

Numerous studies have established a pivotal role for Aβ42 in Alzheimers disease (AD) pathogenesis. In contrast, although Aβ40 is the predominant form of amyloid β (Aβ) produced and accumulates to a variable degree in the human AD brain, its role in AD pathogenesis has not been established. It has generally been assumed that an increase in Aβ40 would accelerate amyloid plaque formation in vivo. We have crossed BRI-Aβ40 mice that selectively express high levels of Aβ40 with both Tg2576 (APPswe, K670N+M671L) mice and BRI-Aβ42A mice expressing Aβ42 selectively and analyzed parenchymal and cerebrovascular Aβ deposition in the bitransgenic mice compared with their singly transgenic littermates. In the bitransgenic mice, the increased steady-state levels of Aβ40 decreased Aβ deposition by 60–90%. These results demonstrate that Aβ42 and Aβ40 have opposing effects on amyloid deposition: Aβ42 promotes amyloid deposition but Aβ40 inhibits it. In addition, increasing Aβ40 levels protected BRI-Aβ40/Tg2576 mice from the premature-death phenotype observed in Tg2576 mice. The protective properties of Aβ40 with respect to amyloid deposition suggest that strategies that preferentially target Aβ40 may actually worsen the disease course and that selective increases in Aβ40 levels may actually reduce the risk for development of AD.

Anti-Aβ42– and anti-Aβ40–specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model

Accumulation and aggregation of amyloid β peptide 1–42 (Aβ42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Aβ42 versus Aβ40 or total Aβ is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Aβ42 mAb, an anti-Aβ40 mAb, and multiple Aβ1–16 mAbs. We established in vivo binding selectivity of the anti-Aβ42 and anti-Aβ40 mAbs using novel TgBRI-Aβ mice. We then conducted a prevention study in which the anti-Aβ mAbs were administered to young Tg2576 mice, which have no significant Aβ deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Aβ deposits. Anti-Aβ42, anti-Aβ40, and anti-Aβ1–16 mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Aβ42 and anti-Aβ40 mAbs were less effective in attenuating Aβ deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Aβ42 or Aβ40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.

Insights into the mechanisms of action of anti-abeta antibodies in Alzheimer's disease mouse models

A number of hypotheses regarding how anti‐Aβ antibodies alter amyloid deposition have been postulated, yet there is no consensus as to how Aβ immunotherapy works. We have examined the in vivo binding properties, pharmacokinetics, brain penetrance, and alterations in Aβ levels after a single peripheral dose of anti‐Aβ antibodies to both wild‐type (WT) and young non‐Aβ depositing APP and BRI‐Aβ42 mice. The rapid rise in plasma Aβ observed after antibody (Ab) administration is attributable to prolongation of the half‐life of Aβ bound to the Ab. Only a miniscule fraction of Ab enters the brain, and despite dramatic increases in plasma Aβ, we find no evidence that total brain Aβ levels are significantly altered. Surprisingly, cerebral spinal fluid Aβ levels transiently rise, and when Ab:Aβ complex is directly injected into the lateral ventricles of mice, it is rapidly cleared from the brain into the plasma where it remains stable. When viewed in context of daily turnover of Aβ, these data provide a framework to evaluate proposed mechanisms of Aβ attenuation mediated by peripheral administration of an anti‐Aβ monoclonal antibody (mAb) effective in passive immunization paradigm. Such quantitative data suggest that the mAbs are either indirectly enhancing clearance of Aβ or targeting a low abundance aggregation intermediate.—Levites, Y., Smithson, L. A., Price, R. W., Dakin. R. S., Yuan, B., Sierks, M. R., Kim, J., McGowan, E., Reed, D. K., Rosenberry, T. L., Das, P., Golde, T. E. Insights into the mechanisms of action of anti‐Aβ antibodies in Alzheimers disease mouse models. FASEB J. 20, E2002–E2014 (2006)

BRI2 (ITM2b) Inhibits Aβ Deposition In Vivo

Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimers disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1–40 transgenes in APP mouse models. Expression of BRI2-Aβ1–40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo.

Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

BackgroundAlzheimers disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD.ResultsAdeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits.ConclusionThe results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Aβ42 alone is sufficient to initiate Aβ deposition, both Aβ40 and Aβ42 may contribute to cognitive deficits.

Interleukin-1 receptor 1 knockout has no effect on amyloid deposition in Tg2576 mice and does not alter efficacy following Aβ immunotherapy

BackgroundMicroglial activation has been proposed to facilitate clearance of amyloid β protein (Aβ) from the brain following Aβ immunotherapy in amyloid precursor protein (APP) transgenic mice. Interleukin-1 receptor 1 knockout (IL-1 R1-/-) mice are reported to exhibit blunted inflammatory responses to injury. To further define the role of IL-1-mediated inflammatory responses and microglial activation in this paradigm, we examined the efficacy of passive Aβ immunotherapy in Tg2576 mice crossed into the IL-1 R1-/- background. In addition, we examined if loss of IL-1 R1-/- modifies Aβ deposition in the absence of additional manipulations.MethodsWe passively immunized Tg2576 mice crossed into the IL-1 R1-/- background (APP/IL-1 R1-/- mice) with an anti-Aβ1-16 mAb (mAb9, IgG2a) that we previously showed could attenuate Aβ deposition in Tg2576 mice. We also examined whether the IL-1 R1 knockout background modifies Aβ deposition in untreated mice. Biochemical and immunohistochemical Aβ loads and microglial activation was assessed.ResultsPassive immunization with anti-Aβ mAb was effective in reducing plaque load in APP/IL-1 R1-/- mice when the immunization was started prior to significant plaque deposition. Similar to previous studies, immunization was not effective in older APP/IL-1 R1-/- mice or IL-1 R1 sufficient wild type Tg2576 mice. Our analysis of Aβ deposition in the untreated APP/IL-1 R1-/- mice did not show differences on biochemical Aβ loads during normal aging of these mice compared to IL-1 R1 sufficient wild type Tg2576 mice.ConclusionWe find no evidence that the lack of the IL-1 R1 receptor influences either Aβ deposition or the efficacy of passive immunotherapy. Such results are consistent with other studies in Tg2576 mice that suggest microglial activation may not be required for efficacy in passive immunization approaches.

P2-347: BRI2 (ITM2B) inhibits Aβ deposition

one protein located on the endoplasmic reticulum (ER) membrane, precisely at focal contacts between the ER and mitochondria (Hayashi & Su, Cell 131:596, 2007). The protein regulates the activity of different ER proteins, like IP3 receptors or ER stress sensors (GRP78/BiP, PERK, ATF-6). The 1 chaperone has the unique particularity to be sensitive to synthetic ligands, which therefore allow a very focal regulation of intracellular calcium homeostasis at ER and/or mitochondria contacts. As a consequence, 1 activators/agonists have been shown to induce acute modulation of transduction pathways, effective at the behavioral level. 1 Activators are indeed anti-amnesic, antidepressant and neuroprotective compounds. We validate new 1 activator compounds as neuroprotective agents against amyloid toxicity and analyze their mechanism of action. Methods: PRE-084 is a morpholine piperidine derivative acting as a high affinity and selective 1 activator. ANAVEX1-41 is a tetrahydro-furanmethanamine that shows high affinity for M1, M2, M4 muscarinic acetylcholine receptors and 1 protein. Both compounds are potent anti-amnesic drugs alleviating learning impairments observed in mice after the central (i.c.v.) injection of amyloid 25-35 peptide (A 25-35). Results: Central administration of A 25-35 induces within one week histological and biochemical changes, memory deficits, oxidative stress and ER stress in sensitive brain structures (hippocampus, cortex), highly reminiscent of the amyloid toxicity observed in Alzheimer’s disease. A 25-35 also provokes the induction of intracellular pro-apoptotic caspases and Bax-related proteins, markers of the induction of apoptosis. At the morphological level, A 25-35 induces a marked glial (astroglia, microglia) reaction and cell loss quantifiable in pyramidal layers of the hippocampus. Pre-administration of PRE-084 or ANAVEX1-41 prevents significantly all these pathological changes, showing that 1 activators are effective neuroprotectants. Part of the mechanism involves regulation of the expression of activity of IP3 receptors or ER stress sensors, in relation with the massive calcium overload induced by A 25-35. Conclusions: 1 Activators/agonists show potent neuroprotective and putatively disease-modifying activity against amyloid toxicity. Moreover, ANAVEX1-41 is active at 30-100 g/kg i.p., suggesting a cooperative action between muscarinic and 1 targets.