Faculty Bibliography

Deposition of amyloid-beta (Abeta) in form of the senile plaques and in vessel walls is a hallmark of Alzheimer's disease (AD). Apolipoprotein E (apoE) is known to act as a pathological chaperone by increasing the beta-sheet content of Abeta, promoting its fibrillization, toxicity, and deposition in the brain. ApoE binds to residues 12-28 of Abeta. We report in vitro and in vivo data on the blocking of the apoE/Abeta interaction by a synthetic peptide homologues to residues 12-28 of Abeta. To eliminate any residual toxicity and fibrillogenic potential the peptide sequence was altered by replacing a valine in position 18 by a proline (Abeta12-28P). On ELISA Abeta12-28P demonstrates high affinity binding to apoE and in competitive binding experiments inhibits the binding of apoE to Abeta42. Abeta12-28P also reduces the toxicity of Abeta in cell culture, as well as blocking the enhanced fibril formation of Abeta in the presence of apoE4, measured by the Thioflavin-T assay. The in vivo effect of Abeta12-28P was assessed in double transgenic (Tg) APP/PS1 AD mice which received 1mg of Abeta12-28P or placebo three times a week for four weeks. There was an approximately five fold reduction of the total and fibrillar Abeta in treated mice comparing to control (p<0.05). Also, Abeta40 and Abeta42 levels in the brain demonstrated a 40-60% reduction of both species in the total Abeta fraction and in the soluble Abeta fraction in treated mice comparing to controls. No significant titer of anti-Abeta antibodies in treated animals was detected, indicating that the effect of Abeta12-28P on Abeta deposition observed in vivo is not immune mediated. Overall, compounds blocking the interaction between Abeta and its pathological chaperones such as apoE (or alpha1anti-chymotrypsin, perlecan etc.) can be considered as an alternative approach for the treatment of beta-amyloidosis in AD

Presenilin 1 (PS1) mutations have been identified in many pedigrees with early-onset familial Alzheimer's disease (FAD). PS1 mutants are known to influence gamma-secretase action and increase amyloid-beta (Abeta) 1-42 production, but there is also evidence suggesting direct involvement of PS1 in the neuronal pathology of AD. Transgenic (Tg) mice expressing the M146L PS1 mutation, associated with FAD symptom onset in the forties, demonstrate no difference in the total number of neurons (fractionator method) in the CA1 sector of the cornu Ammonis comparing with wild type (wt) animals at two months of age. At the age of 9 months and 22 months PS1 M146L Tg mice demonstrated 20% and 29% neuronal dropout comparing to age-matched controls, respectively (p<0.05). Between 2 months and 22 months old wt animals did not show any significant neuronal loss; however, 22 month old M146L PS1 mice showed a 41% neuronal decline compared to 2 month old controls. PS1 M146L Tg animals also exhibited impaired performance of both learning and retention on the Morris water maze test (p<0.05), but not on locomotor testing comparing to wt mice. We have also analyzed another line of Tg mice expressing a P117L PS1 mutation associated with an onset of disease as early as 23 years. These mice at the age of 6 months demonstrate a 17.9% reduction in the total number of CA1 neurons comparing to wt mice and a 26.5% reduction comparing to mice expressing the wt form of human PS1 (p<0.05). Overall, this data suggest that PS1 mutations are directly involved in neuronal pathology which is age-dependant. This process is unrelated to Abeta deposition since PS1 Tg mice do not develop amyloid plaques

According to the amyloid hypothesis, it is the progressive accumulation of beta-amyloid that leads to a cascade of neurodegenerative processes in Alzheimer's disease (AD). Thus, current strategies for diagnosis and treatment evaluation rely on the ability to accurately quantify beta-amyloid burden. It has previously been shown that beta-amyloid plaques can be imaged using Magnetic Resonance Microscopy (MRM) at 40mum isotropic resolution in ex vivo human samples of the hippocampus. Transgenic (Tg) mice have been generated for research as beta-amyloidosis models. Plaque sizes range can from 5mum to 200mum, with an average diameter of approximately 25mum. In the present study, we used high resolution MRM to explore the feasibility of visualizing beta-amyloid plaque deposits in the brain of Tg2576 mice carrying the Swedish mutation of APP. We obtained T2 weighted 3D whole brain MRM data at 20mum and 25mum isotropic resolution. MRM images were compared with histological data to confirm that the signal seen on MRM corresponded to actual beta-amyloid plaque deposits. We conclude that MRM is a practical and useful assay for imaging beta-amyloid plaques with diameters as small as 20mum. These results will aid in the interpretation of MRI data gathered from in-vivo scans of mice, including scans wherein contrast agents are employed. This MRI technique can be easily applied to whole brain plaque quantification studies and for the purpose of studying treatment strategies using mouse models of AD, and may further be extended to in vivo studies tracking amyloid deposit formation and maturation throughout the animals life span

Amyloid deposition in Alzheimer's disease (AD) occurs many years before cognitive impairment. Brain imaging techniques targeting plaques will have an important diagnostic value and may help in identifying individuals in preclinical stages of AD. Magnetic resonance imaging (MRI) has a much higher resolution than positron enhanced tomography (PET) imaging and, therefore, is a more sensitive method to detect amyloid plaques. In our initial proof-of-concept studies (Magnetic Resonance in Medicine, in press), we utilized Abeta1-40 peptide, labeled with gadolinium or monocrystalline iron oxide nanoparticles (MION). When either of these ligands is injected in vivo systemically with mannitol to transiently open the blood-brain-barrier, we are able to image ex vivo the majority of Abeta plaques in Tg mice. Using Gd labeled Abeta1-40 and in vivo muMRI, we can also detect a substantial percentage of amyloid lesions. There is a high correlation between the numerical density of Abeta plaques detected by muMRI and by immunohistochemistry. Clinical use of Abeta1-40 is not feasible because it may add to the plaque burden. As a safer approach, we are using gadolinium labeled K6Abeta1-30, a non-toxic Abeta derivative with low propensity to form beta-sheet, while maintaining high affinity for Abeta. Our initial findings indicate that this compound has a similar effect as gadolinium labeled Abeta1-40 in allowing in vivo detection of amyloid plaques in Tg mice. We are currently exploring various ways to enhance the uptake of this compound into the brain. This approach may lead to a diagnostic MRI method to detect Abetaplaques in AD patients

Calbindin D(28K) (CB) expression was analyzed in the rat hippocampus following 10-min-cardiac arrest-induced ischemia within a year after reperfusion. In rats examined 3 days after ischemia, CB immunoreactivity disappeared completely from CA1 pyramidal neurons and from most CA2 pyramids. In the stratum granulosum of the dentate gyrus, mossy fibers, and hippocampal interneurons, CB immunoreactivity was preserved, although staining was somewhat paler than that in control rats. A similar pattern of CB immunoreactivity was found in rats sacrificed 14 days and 1 month after cardiac arrest. From the 14th postischemic day, neuronal loss in the stratum pyramidale of CA1 but not in that of CA2 became apparent. The reappearance of CB immunoreactivity in CA1 and CA2 pyramidal neurons was noticed 6 months after ischemia, and the pattern was identical to that observed in animals sacrificed 12 months after the ictus. The prolonged loss and delayed reappearance of CB immunoreactivity in the hippocampus demonstrate that ischemia may induce long-term disturbances of protein expression, which may in turn result in impairment of hippocampal functioning

Using axonal retrograde tracing, combined with morphometric analysis, we compared the distribution and number of claustral neurons projecting to the motor and somatosensory cortical areas in the Wistar rat. Comparable volumes of the retrograde tracer Fluoro-Gold, were injected into the motor or somatosensory cortices. Injections into these areas resulted in labeling of neurons along the entire length of the claustrum. Neurons retrogradely labeled after injection into the motor cortex prevailed in the anterior part of the claustrum, whereas those projecting to the somatosensory cortex predominated in the central part. The mean number of claustral neurons retrogradely labeled after tracer injections into the motor cortex significantly outnumbered that from the somatosensory cortical area (p < 0.01). Similarly, the mean value of the numerical density of the retrogradely labeled neurons was significantly higher for the motor projection zone in the claustrum, than for the somatosensory projection zone (p < 0.001). The contralateral claustral projections, both into the motor and somatosensory cortices, were considerably lower in number than the ipsilateral ones. These findings indicate that: (1) the claustral projections to the various cortical regions seem to be differentiated (2) the distribution of claustral neurons projecting to the motor and somatosensory neocortical areas shows an anteroposterior gradient, (3) the claustrum of the rat appears to be more closely related to the motor than to the somatosensory system, (4) the rat claustrum seems to function more as a satellite than a relay structure in relationship to the cerebral cortex.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes

In Alzheimer's disease (AD), neurofibrillary degeneration of neurons starts in the transentorhinal cortex and spreads in a time-dependent manner to the entorhinal cortex, which provides a major input to the hippocampus--a key structure of the memory system. People with Down's syndrome (DS) develop neurofibrillary changes more than 30 years earlier than those with sporadic AD. To characterize AD-related pathology in the entorhinal cortex in DS, we examined seven subjects with DS of 60-74 years of age who died in the end stage of AD, and four age-matched control subjects. The volume of the entorhinal cortex in brains of subjects with DS was 42% less than that in control cases; however, the total number of neurons free of neurofibrillary changes was reduced in DS by 90%: from 9,619,000 +/- 914,000 (mean +/- standard deviation) to 932,000 +/- 504,000. The presence of 2,488,000 +/- 544,000 neurofibrillary tangles in the entorhinal cortex of people with DS, the prevalence of end-stage tangles, and the significant negative correlation between the total number of intact neurons and the percentage of neurons with neurofibrillary changes indicate that neurofibrillary degeneration is a major cause of neuronal loss in the entorhinal cortex of people with DS. The relatively low amyloid load (7 +/- 1%) and lack of correlation between the amyloid load and the volumetric or neuronal loss suggest that the contribution of beta-amyloid to neuronal loss in the entorhinal cortex is unsubstantial