Cynthia DeLong

Immunization reverses memory deficits without reducing brain Aβ burden in Alzheimer's disease model

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid β-peptide (Aβ), increases plasma concentrations of Aβ and reduces Aβ burden in the PDAPP transgenic mouse model of Alzheimers disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Aβ burden. We also found that an Aβ/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Aβ monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Aβ species.

Human APOE Isoform-Dependent Effects on Brain β-Amyloid Levels in PDAPP Transgenic Mice

To investigate the role of human apolipoprotein E (apoE) on Aβ deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and Aβ peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain Aβ and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain Aβ deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of Aβ42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain Aβ burden, and the majority of apoE was associated with Aβ. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 < E3 ≪ E2), resulting in early and dramatic apoE isoform-dependent effects on brain Aβ levels (E4 ≫ E3 > E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimers disease.

Macrophage-Mediated Degradation of β-Amyloid via an Apolipoprotein E Isoform-Dependent Mechanism

Recent studies suggest that bone marrow-derived macrophages can effectively reduce β-amyloid (Aβ) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Aβ, we cultured murine macrophages on top of Aβ plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP717V>F mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Aβ in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Aβ degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Aβ than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Aβ was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Aβ-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Aβ degradation by macrophages. These apoE isoform-dependent effects of macrophages on Aβ degradation suggest a novel “peripheral” mechanism for Aβ clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimers disease.

Emergence of a seizure phenotype in aged apolipoprotein epsilon 4 targeted replacement mice.

The apolipoprotein ε4 allele is the strongest genetic risk factor for late-onset Alzheimers disease (AD) and is associated with earlier age of onset. The incidence of spontaneous seizures has been reported to be increased in sporadic AD as well as in the early onset autosomal dominant forms of AD. We now report the emergence of a seizure phenotype in aged apolipoprotein E4 (apoE4) targeted replacement (TR) mice but not in age-matched apoE2 TR or apoE3 TR mice. Tonic-clonic seizures developed spontaneously after 5 months of age in apoE4 TR mice and are triggered by mild stress. Female mice had increased seizure penetrance compared to male mice, but had slightly reduced overall seizure severity. The majority of seizures were characterized by head and neck jerks, but 25% of aged apoE4 TR mice had more severe tonic-clonic seizures which occasionally progressed to tonic extension and death. Aged apoE4 TR mice progressed through pentylenetetrazol-induced seizure stages more rapidly than did apoE3 TR and apoE2 TR mice. Electroencephalographic (EEG) recordings revealed more frequent bursts of synchronous theta activity in the hippocampus of apoE4 TR mice than in apoE2 TR or apoE3 TR mice. Cortical EEG recordings also revealed sharp spikes and other abnormalities in apoE4 TR mice. Taken together, these findings demonstrate the emergence of an age-dependent seizure phenotype in old apoE4 TR mice in the absence of human amyloid-β peptide (Aβ) overexpression, suggesting increased central nervous system neural network excitability.

Statistical aspects of quantitative image analysis of β-amyloid in the APPV717F transgenic mouse model of Alzheimer's disease

Cerebral beta-amyloidosis is a central part of the neuropathology of Alzheimers disease (AD). Quantitation of beta-amyloid plaques in the human AD brain, and in animal models of AD, is an important study endpoint in AD research. Methodologic approaches to the measurement of beta-amyloid in the brain vary between investigators, and these differences affect outcome measures. Here, one quantitative approach to the measurement of beta-amyloid plaques in brain sections was analyzed for sources of variability due to sampling. Brain tissue was from homozygous APP(V717F) transgenic male mice. Sampling variables were at the mouse and microscopic slide and field levels. Results indicated that phenotypic variability in the mouse sample population was the largest contributor to the standard error of the analyses. Within each mouse, variability between slides or between fields within slides had smaller effects on the error of the analyses. Therefore, when designing studies of adequate power, in this and in other similar models of cerebral beta-amyloidosis, sufficient numbers of mice per group must be included in order for change in mean plaque burden attributable to an experimental variable to outweigh phenotypic variability.

CHARACTERIZATION OF AMINO TERMINAL TRUNCATIONS OF PLAQUE-ASSOCIATED Aβ AFTER COMBINATION BACE INHIBITOR AND ANTIBODY THERAPY IN AGED PDAPP MICE

TRUNCATIONS OF PLAQUE-ASSOCIATED Ab AFTER COMBINATION BACE INHIBITOR AND ANTIBODY THERAPY IN AGED PDAPP MICE Margaret Racke, John Tzaferis, Justin Hole, Feng Liu, Cynthia DeLong, Patrick May, Michael Hutton, Christer Nordstedt, Ronald DeMattos, Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Company, Ltd., Windlesham, United Kingdom; 5 Eli Lilly and Company, Indianapolis, Indiana, United States. Contact e-mail: racke_margaret@ lilly.com