Treatment effects in a transgenic mouse model of Alzheimer's disease: a magnetic resonance spectroscopy study after passive immunization.

Despite the enormous public health impact of Alzheimer's disease (AD), no disease-modifying treatment has yet been proven to be efficacious in humans. A rate-limiting step in the discovery of potential therapies for humans is the absence of efficient non-invasive methods of evaluating drugs in animal models of disease. Magnetic resonance spectroscopy (MRS) provides a non-invasive way to evaluate the animals at baseline, at the end of treatment, and serially to better understand treatment effects. In this study, MRS was assessed as potential outcome measure for detecting disease modification in a transgenic mouse model of AD. Passive immunization with two different antibodies, which have been previously shown to reduce plaque accumulation in transgenic AD mice, was used as intervention. Treatment effects were detected by MRS, and the most striking finding was attenuation of myo-inositol (mIns) increases in APP-PS1 mice with both treatments. Additionally, a dose-dependent effect was observed with one of the treatments for mIns. MRS appears to be a valid in vivo measure of anti-Aß therapeutic efficacy in pre-clinical studies. Because it is noninvasive, and can detect treatment effects, use of MRS-based endpoints could substantially accelerate drug discovery.

Regional differences in MRI detection of amyloid plaques in AD transgenic mouse brain.

Our laboratory and others have reported the ability to detect individual Alzheimer's disease (AD) amyloid plaques in transgenic mouse brain in vivo by magnetic resonance imaging (MRI). Since amyloid plaques contain iron, most MRI studies attempting to detect plaques in AD transgenic mouse brain have employed techniques that exploit the paramagnetic effect of iron and have had mixed results. In the present study, using five-way anatomic spatial coregistration of MR images with three different histological techniques, properties of amyloid plaques in AD transgenic mouse brain were revealed that may explain their variable visibility in gradient- and spin-echo MR images. The results demonstrate differences in the visibility of plaques in the cortex and hippocampus, compared to plaques in the thalamus, by the different MRI sequences. All plaques were equally detectable by T(2)SE, while only thalamic plaques were reliably detectable by T(2)*GE pulse sequences. Histology revealed that cortical/hippocampal plaques have low levels of iron while thalamic plaques have very high levels. However, the paramagnetic effect of iron does not appear to be the sole factor leading to the rapid decay of transverse magnetization (short T(2)) in cortical/hippocampal plaques. Accordingly, MRI methods that rely less on iron magnetic susceptibility effect may be more successful for eventual human AD plaque MR imaging, particularly since human AD plaques more closely resemble the cortical and hippocampal plaques of AD transgenic mice than thalamic plaques.

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 Alzheimer's 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 Alzheimer's disease.

Therapeutic benefit of polyamine-modified catalase as a scavenger of hydrogen peroxide and nitric oxide in familial amyotrophic lateral sclerosis transgenics.

Continuous subcutaneous administration of polyamine-modified catalase that has increased permeability at the blood-brain barrier showed both a highly significant delay in onset and an increase in survival in a transgenic mouse model of familial amyotrophic lateral sclerosis having a point mutation in the gene encoding copper/zinc superoxide dismutase. These results suggest that hydrogen peroxide-mediated oxidative stress with subsequent free radical damage involving nitric oxide and possibly hydroxyl radicals in motor neurons may be the culprit in familial amyotrophic lateral sclerosis.

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 Alzheimer's 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.

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-beta peptide (Abeta), 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 Abeta 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 Abeta 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.

Putrescine-modified catalase with preserved enzymatic activity exhibits increased permeability at the blood-nerve and blood-brain barriers.

Much evidence exists in support of the hypothesis that free radicals contribute to the pathogenesis of several neurodegenerative disorders and that mechanisms of free radical generation occur both intracellularly and extracellularly. Previous studies in this laboratory have shown that covalent modification of growth factors and antioxidant enzymes with the naturally occurring polyamine, putrescine, increases their permeability at the blood-nerve and blood-brain barriers (BNB and BBB), but does not significantly inhibit bioactivity. Furthermore, putrescine-modified superoxide dismutase (SOD) was shown to reduce neurodegeneration in a rat model of global cerebral ischemia. The purpose of the present study was to modify the antioxidant enzyme, catalase (CAT), with putrescine (PUT) at carboxylic acid groups whose ionization, and hence reactivity, was controlled with pH and investigate the effects on permeability and enzymatic activity. Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT and 137% compared to lyophilized CAT. The results of this study indicate that modification of CAT with putrescine increases its permeability while preserving enzymatic activity. PUT-SOD administered in combination with PUT-CAT may eliminate both the superoxide radical and the H2O2 produced from the dismutation of superoxide, respectively, and thus prevent the formation of hydroxyl radicals. This combination may exhibit increased neuroprotective effects, compared to native enzymes, following systemic administration for the treatment of free radical associated neurodegenerative disorders.

Postischemic, systemic administration of polyamine-modified superoxide dismutase reduces hippocampal CA1 neurodegeneration in rat global cerebral ischemia.

Antioxidant enzymes such as superoxide dismutase (SOD) have shown neuroprotective effects in animal models of cerebral ischemia, but only at very high doses. Modifications to increase the plasma half-life or blood-brain barrier (BBB) permeability of SOD have resulted in limited neuroprotective effects. No one has demonstrated neuroprotection with postischemic administration. The specific aim of the present study was to administer systemically a polyamine-modified SOD, having increased BBB permeability and preserved enzymatic activity, following global cerebral ischemia in rats and analyze the effects on the selective vulnerability of CA1 hippocampal neurons. Following 12 min of four-vessel occlusion, global cerebral ischemia, male Wistar rats were dosed (i.v.) with either saline, native SOD (5000 U/kg), polyamine-modified SOD (5000 U/kg), or enzymatically inactive, polyamine-modified SOD (2.1 mg/kg) twice daily for 3 days. Neuroprotective effects on hippocampal CA1 neurons were assessed using standard histological methods. Saline-treated animals had very few remaining CA1 neurons (1.44 +/- 0.60 neurons/reticle; x +/- S.E.M.) compared to sham rats (58.57 +/- 0.69). Native (10.38 +/- 2.96) or inactive, polyamine-modified SOD (7.32 +/- 2.68) did not show significant neuroprotective effects. Polyamine-modified SOD, however, resulted in the survival of significantly more CA1 neurons (24.61 +/- 5.90; P < 0.01). Postischemic, systemic administration of polyamine-modified SOD, having increased BBB permeability and preserved enzymatic activity, significantly reduced hippocampal CA1 neuron loss following global cerebral ischemia. Similar modification of other antioxidant enzymes and neurotrophic factors with polyamines may provide a useful technique for the systemic delivery of therapeutic proteins across the BBB for the treatment of stroke and other neurodegenerative disorders.

Profiling of endogenous peptides as a tool for studying development and neurological disease.

The use of a method to follow changes in endogenous peptide production, as they occur in biological studies, is an excellent complement to other molecular techniques. It has the unique ability to characterize peptides that have been produced from protein precursors, and instrumentation is available that provides high resolution peptide separations that are quantitative, sensitive, and amenable to automation. All tissues express a large number of peptide species that can be visualized, or profiled, on chromatographic separations using reverse-phase high-performance liquid chromatography. This large number of peptides offers many potential molecules that can be used to identify biological mechanisms associated with experimental paradigms. Peptide analysis has been used successfully in many types of studies. In this review, we outline our experience in using peptides as biological markers and provide a description of the evolution of peptide profiling in our laboratories. Peptide expression has been used in studies ranging from how brain regions develop to identifying changes in disease processes including Alzheimer's disease and models of stroke. Some of the findings provided by these studies have been new pathways of peptide processing and the identification of accelerated proteolysis on proteins such as hemoglobin as a function of Alzheimer's disease and brain insult. Peptide profiling has also proven to be an excellent technique for studying many well-known nervous system proteins including calmodulin, PEP-19, myelin basic protein, cytoskeletal proteins, and others. It is the purpose of this review to describe our experience using the technique and to highlight improvements that have added to the power of the approach. Peptide analysis and the expansion in the instrumentation that can detect peptides will no doubt make these types of studies a powerful addition to our molecular armamentarium for conducting biological studies.

Camstatins are peptide antagonists of calmodulin based upon a conserved structural motif in PEP-19, neurogranin, and neuromodulin.

Unbridled increases in intracellular ionized calcium can result in neuronal damage and death. Since many of the deleterious effects of calcium are mediated by calmodulin, we have sought to identify neuronal proteins that inhibit activation of this ubiquitous protein. PEP-19 is a 7.6-kDa neuron-specific protein, which contains a motif similar to the calmodulin binding domains of neuromodulin (GAP-43) and neurogranin (RC3). Here we show that PEP-19 binds calmodulin in an analogous calcium-independent manner with an apparent Kd near 1.2 microM. Furthermore, using the calmodulin-dependent enzyme neuronal nitric oxide synthase, we demonstrate that native PEP-19 is also an antagonist of enzyme activity. Based on the PEP-19 sequence, a series of peptide calmodulin antagonists termed camstatins were synthesized. These analogs define the minimally active domain of PEP-19 and provide a structure/activity relationship for calmodulin antagonism. There was a positive correlation between the binding affinities of the camstatins for calmodulin and their potencies as neuronal nitric oxide synthase inhibitors. Despite the similar IQ motif in PEP-19 and neuromodulin or neurogranin, PEP-19 was not a substrate for protein kinase C. The properties of PEP-19 suggest that it could fulfill a role in neuroprotection.

Selective vulnerability and early progression of hippocampal CA1 pyramidal cell degeneration and GFAP-positive astrocyte reactivity in the rat four-vessel occlusion model of transient global ischemia.

Selective, delayed-onset vulnerability of hippocampal CA1 pyramidal cells has been reported as a unique phenomenon in man and the rat four-vessel occlusion (4-VO) model of global ischemia. This has become of great interest for clarification of CA1 pathophysiology and pharmacological intervention after global ischemia. Studies of pathophysiology and pharmacotherapy appear to be impeded by variability in specific criteria and duration of 4-VO ischemia for producing selective CA1 and differential CA1-CA3 damage. The goals of this study were to: (1) develop specific criteria for 4-VO ischemia to ensure selective, bilaterally symmetrical CA1 pyramidal cell damage, (2) examine the effects of 15 min of ischemia on concomitant CA1 cell necrosis and presence of remaining and/or "viable" neurons postischemia, (3) compare 15 and 30 min of ischemia on differential vulnerability of CA1-CA3 subfields, and (4) evaluate the effects of 15 min of ischemia on CA1 pyramidal cell necrosis and glial fibrillary acidic protein (GFAP)-positive astrocyte reactivity in CA1. After 15 min of ischemia, hippocampal pyramidal cell damage was well delineated, with CA1 severely damaged, but leaving CA3 virtually intact. In contrast, 30 min of ischemia produced severe CA1 and less severe CA3 necrosis. Histological evaluations across Days 1, 3, 6, and 14 indicated a significant delayed onset of CA1-CA3 cell necrosis by Day 3. Counting of remaining cells indicated a detectable loss of some large pyramidal neurons even 1 day after ischemia. Compared to controls, there was a differential increase in GFAP-positive astrocytes in CA1-CA3 after ischemia. The results provided quantitative data on the effects of specific 4-VO criteria and durations on: (1) selective CA1 cell necrosis, (2) differential CA1-CA3 cell vulnerability, (3) presence of postischemic remaining and/or viable neurons, and (4) prospect of a "therapeutic window" for pharmacological treatment of CA1 neuronal injury.