Thomas M. Wengenack

Molecular targeting of Alzheimer's amyloid plaques for contrast-enhanced magnetic resonance imaging.

Smart molecular probes for both diagnostic and therapeutic purposes are expected to provide significant advances in clinical medicine and biomedical research. We describe such a probe that targets beta-amyloid plaques of Alzheimers disease and is detectable by magnetic resonance imaging (MRI) because of contrast imparted by gadolinium labeling. Three properties essential for contrast enhancement of beta-amyloid plaques on MRI exist in this smart molecular probe, putrescine-gadolinium-amyloid-beta peptide: (1) transport across the blood-brain barrier following intravenous injection conferred by the polyamine moiety, (2) binding to plaques with molecular specificity by putrescine-amyloid-beta, and (3) magnetic resonance imaging contrast by gadolinium. MRI was performed on ex vivo tissue specimens at 7 T at a spatial resolution approximating plaque size (62.5 microm(3)), in order to prove the concept that the probe, when administered intravenously, can selectively enhance plaques. The plaque-to-background tissue contrast-to-noise ratio, which was precisely correlated with histologically stained plaques, was enhanced more than nine-fold in regions of cortex and hippocampus following intravenous administration of this probe in AD transgenic mice. Continuing engineering efforts to improve spatial resolution are underway in MRI, which may enable in vivo imaging at the resolution of individual plaques with this or similar contrast probes. This could enable early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.

In vivo visualization of Alzheimer's amyloid plaques by magnetic resonance imaging in transgenic mice without a contrast agent.

One of the cardinal pathologic features of Alzheimers disease (AD) is the formation of senile, or amyloid, plaques. Transgenic mice have been developed that express one or more of the genes responsible for familial AD in humans. Doubly transgenic mice develop “human‐like” plaques, providing a mechanism to study amyloid plaque biology in a controlled manner. Imaging of labeled plaques has been accomplished with other modalities, but only MRI has sufficient spatial and contrast resolution to visualize individual plaques noninvasively. Methods to optimize visualization of plaques in vivo in transgenic mice at 9.4 T using a spin echo sequence based on adiabatic pulses are described. Preliminary results indicate that a spin echo acquisition more accurately reflects plaque size, while a T2* weighted gradient echo sequence reflects plaque iron content, not plaque size. In vivo MRI–ex vivo MRI–in vitro histologic correlations are provided. Histologically verified plaques as small as 50 μm in diameter were visualized in living animals. To our knowledge this work represents the first demonstration of noninvasive in vivo visualization of individual AD plaques without the use of a contrast agent. Magn Reson Med 52:1263–1271, 2004.

In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer's Transgenic Mice

The ability to detect individual Alzheimers amyloid plaques in vivo by magnetic resonance microimaging (MRI) should improve diagnosis and also accelerate discovery of effective therapeutic agents for Alzheimers disease (AD). Here, we perform in vivo and ex vivo MRI on double transgenic AD mice as well as wild-type mice at varying ages and correlate these with thioflavin-S and iron staining histology. Quantitative counts of individual plaques on MRI increase with age and correlate with histologically determined plaque burden. Plaques 20 μm in diameter can be detected in AD mice as young as 3 months of age with ex vivo MRI. Plaques 35 μm in diameter can be detected by 9 months of age with in vivo MRI. In vivo MRI of individual Alzheimers amyloid plaques provides a noninvasive estimate of plaque burden in transgenic AD mice that might be useful in assessing the efficacy of amyloid reduction therapies.

Defects in Mitochondrial Dynamics and Metabolomic Signatures of Evolving Energetic Stress in Mouse Models of Familial Alzheimer's Disease

Background The identification of early mechanisms underlying Alzheimers Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics. Methods and Findings We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans. Conclusions Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD.

Targeting Alzheimer amyloid plaques in vivo

The only definitive diagnosis for Alzheimer disease (AD) at present is postmortem observation of neuritic plaques and neurofibrillary tangles in brain sections. Radiolabeled amyloid-β peptide (Aβ), which has been shown to label neuritic plaques in vitro, therefore could provide a diagnostic tool if it also labels neuritic plaques in vivo following intravenous injection. In this study, we show that the permeability of Aβ at the blood–brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine. We also show that, following intravenous injection, radiolabeled, putrescine-modified Aβ labels amyloid deposits in vivo in a transgenic mouse model of AD, as well as in vitro in human AD brain sections. This technology, when applied to humans, may be used to detect plaques in vivo, allowing early diagnosis of the disease and therapeutic intervention before cognitive decline occurs.

Permeability of proteins at the blood-brain barrier in the normal adult mouse and double transgenic mouse model of Alzheimer's disease

The permeability of albumin, insulin, and human A beta 1--40 at the blood-brain barrier (BBB) was determined in the normal adult mouse (B6/SJL) and in the double transgenic Alzheimer mouse (APP, PS1) by using an I.V. bolus injection technique to quantify the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (V(p)) occupied by the protein in the blood vessels of different brain regions using a second aliquot of the same protein radiolabeled with a different isotope of iodine ((125)I vs (131)I) as a vascular space marker. This technology for quantifying BBB permeability of proteins was adapted from the rat to the mouse and involved catheterizing the femoral artery and vein of the mouse instead of the brachial artery and vein as for the rat. Because of the smaller blood volume in the mouse, serial sampling (20 microl) of blood from the femoral artery of the mouse was performed and directly TCA precipitated to generate a whole blood washout curve for the intact protein. When similar blood sampling techniques were used in the rat, the PS values for albumin and insulin at the BBB were similar in these two species. In the double transgenic mouse, the V(p) values for albumin were significantly increased 1.4- to 1.6-fold in five of six brain regions compared to the normal adult mouse, which indicated increased adherence of albumin to vessel walls. As a result, the PS values were significantly decreased, from 1.4- to 3.2-fold, which likely reflected decreased transport of albumin by passive diffusion. In contrast, insulin, which is taken up into the brain by a receptor-mediated transport mechanism at the BBB, showed no significant difference in the V(p) values but a significant increase in the PS values in four of six brain regions. This suggests a compensatory mechanism in the Alzheimers transgenic brain whereby there is an increased permeability to insulin at the BBB. Surprisingly, there was no significant difference in the V(p) or PS values for human A beta 1--40 at the BBB in the double transgenic Alzheimer mouse at 24, 32, or 52 weeks of age, when there is both significant A beta levels in the plasma and amyloid burden in the brains of these animals. These data suggest that there is not an alteration in permeability to human A beta 1--40 at the BBB with increasing amyloid burden in the double transgenic Alzheimer mouse. Although these observations suggest structural alterations at the BBB, they do not support the concept of extensive BBB damage with substantial increases in BBB permeability in Alzheimers disease.

Quantitative histological analysis of amyloid deposition in Alzheimer's double transgenic mouse brain.

The development of transgenic mice has created new opportunities for the generation of animal models of human neurodegenerative diseases where previously there was no animal counterpart. The first successful transgenic mouse model of Alzheimers disease expressed increased levels of mutant human amyloid precursor protein, exhibiting neuritic-type amyloid deposits and behavioral deficits at six to nine months of age. More recently, it was shown that transgenic mice expressing both mutant human amyloid precursor protein and presenilin 1 exhibit neuritic-type amyloid deposits and behavioral deficits in as little as 12 weeks. This accelerated Alzheimer phenotype greatly reduces the time necessary to conduct preclinical drug trials, as well as animal housing costs. The purpose of this study was to quantify the deposition of amyloid in five regions of the cortex and two regions of the hippocampus of transgenic mice expressing amyloid precursor protein (K670N, M671L) and presenilin 1 (M146L) mutations at various ages, using quantitative methods of confocal laser scanning microscopy and image analysis. Amyloid burden, expressed as the percentage area occupied by thioflavin S-positive amyloid deposits, increased an average of 179-fold from 12 to 54 weeks of age (0.02+/-0.01% to 3.57+/-0.29%, mean+/-S.E.M., respectively) in five regions of the cortex and two of the hippocampus. This was a function of increases in both deposit number and size. This transgenic mouse provides an ideal animal model for evaluating the efficacy of potential therapeutic agents aimed at reducing amyloid deposition, such as inhibitors of amyloid fibril formation or secretase inhibitors.

Magnetic Resonance Imaging of Alzheimer's Pathology in the Brains of Living Transgenic Mice: A New Tool in Alzheimer's Disease Research

Alzheimers disease (AD) is the most common cause of dementia in the elderly. Cardinal pathologic features of AD are amyloid plaques and neurofibrillary tangles, and most in the field believe that the initiating events ultimately leading to clinical AD center on disordered metabolism of amyloid beta protein. Mouse models of AD have been created by inserting one or more human mutations associated with disordered amyloid metabolism and that cause early onset familial AD into the mouse genome. Human-like amyloid plaque formation increases dramatically with age in these transgenic mice. Amyloid reduction in humans is a major therapeutic objective, and AD transgenic mice allow controlled study of this biology. Recent work has shown that amyloid plaques as small as 35 μm can be detected using in vivo magnetic resonance microimaging (MRMI) at high magnetic field (9.4 T). In addition, age-dependent changes in metabolite concentration analogous to those that have been identified in human AD patients can be detected in these transgenic mice using single-voxel 1H magnetic resonance spectroscopy (1H MRS) at high magnetic field. These MR-based techniques provide a new set of tools to the scientific community engaged in studying the biology of AD in transgenic models of the disease. For example, an obvious application is evaluating therapeutic modification of disease progression. Toward the end of this review, the authors include results from a pilot study demonstrating feasibility of using MRMI to detect therapeutic modification of plaque progression in AD transgenic mice.

In vivo targeting of antibody fragments to the nervous system for Alzheimer’s disease immunotherapy and molecular imaging of amyloid plaques

Targeting therapeutic or diagnostic proteins to the nervous system is limited by the presence of the blood–brain barrier. We report that a F(ab′)2 fragment of a monoclonal antibody against fibrillar human Aβ42 that is polyamine (p)‐modified has increased permeability at the blood–brain barrier, comparable binding to the antigen, and comparable in vitro binding to amyloid plaques in Alzheimer’s disease (AD) transgenic mouse brain sections. Intravenous injection of the pF(ab′)24.1 in the AD transgenic mouse demonstrated efficient targeting to amyloid plaques throughout the brain, whereas the unmodified fragment did not. Removal of the Fc portion of this antibody derivative will minimize the inflammatory response and cerebral hemorrhaging associated with passive immunization and provide increased therapeutic potential for treating AD. Coupling contrast agents/radioisotopes might facilitate the molecular imaging of amyloid plaques with magnetic resonance imaging/positron emission tomography. The efficient delivery of immunoglobulin G fragments may also have important applications to other neurodegenerative disorders or for the generalized targeting of nervous system antigens.

Activation of programmed cell death markers in ventral horn motor neurons during early presymptomatic stages of amyotrophic lateral sclerosis in a transgenic mouse model

The identification of the pathogenic mechanism of selective motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) may lead to the development of new therapies to halt or slow the disease course. A novel, MN-specific, Fas-mediated programmed cell death (PCD) pathway has been reported in MNs which involves the activation of p38 MAP kinase (phospho-p38) and neuronal nitric oxide synthase (nNOS). PCD was found to be exacerbated in MNs expressing ALS-linked superoxide dismutase (SOD) mutations. Because this MN-specific pathway was investigated in vitro, we performed an in vivo study to evaluate its potential involvement in MN loss in the lumbar region of spinal cord of mutant SOD transgenic (G93A) mice. Compared to nontransgenic littermates, we found significant increases in the numbers of immunopositive ventral horn MNs of G93A mice as young as 60 days of age for several constituents of this putative PCD pathway, including phospho-p38, nNOS, phospho-ASK1 MAP kinase kinase, and active caspase-3. This study provides in vivo evidence of an MN-specific PCD pathway that may be a pathogenic mechanism of ALS and may be activated very early in the disease process, well before clinical symptoms are evident (200 days). These findings suggest that early diagnosis and therapeutic intervention may be critical for the successful treatment of the disease. These enzymes may provide new markers for earlier diagnosis of ALS and new molecular targets for therapeutic intervention.