Eleanor Drummond

Chemical Fluorescent Probe for Detection of Aβ Oligomers

Aggregation of amyloid β-peptide (Aβ) is implicated in the pathology of Alzheimers disease (AD), with the soluble, Aβ oligomeric species thought to be the critical pathological species. Identification and characterization of intermediate species formed during the aggregation process is crucial to the understanding of the mechanisms by which oligomeric species mediate neuronal toxicity and following disease progression. Probing these species proved to be extremely challenging, as evident by the lack of reliable sensors, due to their heterogeneous and transient nature. We describe here an oligomer-specific fluorescent chemical probe, BoDipy-Oligomer (BD-Oligo), developed through the use of the diversity-oriented fluorescent library approach (DOFLA) and high-content, imaging-based screening. This probe enables dynamic oligomer monitoring during fibrillogenesis in vitro and shows in vivo Aβ oligomers staining possibility in the AD mice model.

Alzheimer’s disease: experimental models and reality

Experimental models of Alzheimer’s disease (AD) are critical to gaining a better understanding of pathogenesis and to assess the potential of novel therapeutic approaches. The most commonly used experimental animal models are transgenic mice that overexpress human genes associated with familial AD (FAD) that result in the formation of amyloid plaques. However, AD is defined by the presence and interplay of both amyloid plaques and neurofibrillary tangle pathology. The track record of success in AD clinical trials thus far has been very poor. In part, this high failure rate has been related to the premature translation of highly successful results in animal models that mirror only limited aspects of AD pathology to humans. A greater understanding of the strengths and weakness of each of the various models and the use of more than one model to evaluate potential therapies would help enhance the success of therapy translation from preclinical studies to patients. In this review, we summarize the pathological features and limitations of the major experimental models of AD, including transgenic mice, transgenic rats, various physiological models of sporadic AD and in vitro human cell culture models.

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer's disease brain tissue.

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer’s disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer’s disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

Developing therapeutic vaccines against Alzheimer's disease.

ABSTRACT Alzheimer’s disease (AD) is the most common form of dementia worldwide. It is characterized by an imbalance between the production and clearance of amyloid β (Aβ) and tau proteins. In AD these normal proteins accumulate, leading to aggregation and a conformational change forming oligomeric and fibrillary species with a high β-sheet content. Active and passive immunotherapeutic approaches result in dramatic reduction of Aβ pathology in AD animal models. However, there is much more limited evidence in human studies of significant clinical benefits from these strategies and it is becoming apparent that they may only be effective very early in AD. Vaccination targeting only tau pathology has shown benefits in some mouse studies but human studies are limited. Greater therapeutic efficacy for the next generation of vaccine approaches will likely benefit from specifically targeting the most toxic species of Aβ and tau, ideally simultaneously.

Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer’s Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics

Here, we describe a new method that allows localized proteomics of amyloid plaques and neurofibrillary tangles (NFTs), which are the two pathological hallmarks of Alzheimers disease (AD). Amyloid plaques and NFTs are visualized using immunohistochemistry and microdissected from archived, formalin-fixed paraffin-embedded (FFPE) human tissue samples using laser-capture microdissection. The majority of human tissue specimens are FFPE; hence the use of this type of tissue is a particular advantage of this technique. Microdissected tissue samples are solubilized with formic acid and deparaffinized, reduced, alkylated, proteolytically digested, and desalted. The resulting protein content of plaques and NFTs is determined using label-free quantitative LC-MS. This results in the unbiased and simultaneous quantification of ~900 proteins in plaques and ~500 proteins in NFTs. This approach permits downstream pathway and network analysis, hence providing a comprehensive overview of pathological protein accumulation found in neuropathological features in AD.

Recent advancements toward therapeutic vaccines against alzheimer’s disease

ABSTRACT Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by protein aggregates of amyloid β (Aβ) and tau. These proteins have normal physiological functions, but in AD, they undergo a conformational change and aggregate as toxic oligomeric and fibrillar species with a high β-sheet content. Areas covered: Active and passive immunotherapeutic approaches are among the most attractive methods for targeting misfolded Aβ and tau. Promising preclinical testing of various immunotherapeutic approaches has yet to translate to cognitive benefits in human clinical trials. Knowledge gained from these past failures has led to the development of second-generation Aβ-active immunotherapies, anti-Aβ monoclonal antibodies targeting a wide array of Aβ conformations, and to a number of immunotherapies targeting pathological tau. This review covers the more recent advances in vaccine development for AD from 2016 to present. Expert commentary: Due to the complex pathophysiology of AD, greatest clinical efficacy will most likely be achieved by concurrently targeting the most toxic forms of both Aβ and tau.

Vascular insufficiency, not inflammation, contributes to chronic gliosis in a rat CNS transplantation model

PURPOSE There is considerable variability in the extent and nature of the glial response to injury and neurodegeneration. Transplantation of fetal cortical tissue onto the brain of neonatal host rats or mice results in region-specific changes dependent on where the fetal tissue is placed. These changes include chronic astrocytic and microglial gliosis, oxidative stress, and altered metabolism of a number of proteins associated with the pathogenesis of Alzheimers disease. Such changes are only observed in heterotopic (cortex-to-midbrain) grafts and are not observed in homotopic cortex-to-cortex grafts. We investigated two possible triggers for the region-specific gliosis observed in our transplant model hypothesizing that either i) poor vascularization and lack of blood brain barrier integrity or ii) an inflammatory response initiated by the transplantation process, contributed to establishing chronic pathological changes. METHODS We analyzed the time course of neovascularization, blood brain barrier permeability and inflammation using a combination of immunohistochemistry, enzyme-linked immunosorbant assay and Evans blue dye extravasation techniques. RESULTS Blood brain barrier permeability and altered neovascularization occurred prior to the onset of gliosis in heterotopic grafts. CONCLUSION These data suggest that ischemic conditions and blood brain barrier damage can be a primary mechanism that initiates chronic gliosis and associated inflammatory changes in central nervous system tissue.

ALTERED PROTEIN EXPRESSION IN AMYLOID PLAQUES IN RAPIDLY PROGRESSIVE ALZHEIMER’S DISEASE

tissue homogenates with exogenous Ab1-42 under multiple conditions. Samples were analyzed using acid urea gels followed by Western blotting. Results:The PDAPP mice study revealed Ab42 to Ab40 conversion over time reaching equilibrium by 72hr. Acid urea gel analyses demonstrated that over half of the Ab142 peptide administered was converted to Ab1-40. Furthermore, ELISA results from rat studies showed similar conversion rates from Ab42 to Ab40 regardless of the route of administration, centrally or peripherally. Ex-vivo studies using rat tissue homogenates incubated with exogenous Ab1-42 peptide also exhibited Ab conversion. This conversion was present in all tissues tested, cortex, kidney, liver, pancreas, and spleen, and was exacerbated when the pH was lowered to pH5 from pH7. The rate of conversion to Ab40 was diminished when a c-terminal antibody or protease inhibitor was incorporated into the in-vivo/ex-vivo studies. Conclusions: We have identified in-vivo processing of the carboxyl-terminus of Ab in rodents. The extent of in-vivo processing is exacerbated when an Ab antibody extends the half-life of the peptide. The conversion of Ab1-42 to Ab1-40 occurs both centrally and peripherally. Although the potential for this conversion in human is unknown, these results suggest additional biology after secretase liberation of the Ab from the APP may be important for the overall Ab ratios being measured in CNS and periphery.

TARGETING THE SHARED PATHOLOGICAL CONFORMERS OF BOTH Aβ AND HYPERPHOSPHYLATED TAU WITH A CONFORMATIONALLY SELECTIVE MONOCLONAL ANTIBODY

Background: Pathological tau aggregation leading to filamentous tau inclusions characterizes neurodegenerative tauopathies such as Alzheimer’s Disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau aggregation is linked with clinical symptoms and probably represents the mediator of neurodegeneration. At the moment there is no cure for these diseases and existing treatments are only of palliative nature. Methods: Transgenic mice that overexpress human P301S tau mutation resemble many neuropathological features of human tauopathies in combination with behavioral deficits and increased mortality. We tested the effect of the small molecule anle138b as an anti-aggregative drug on the neuropathology and behavioral deficits in P301S mice. The compound was administered from weaning until death. Results: Here we show that reducing the amount of tau aggregates using the compound anle138b ameliorates disease symptoms in a mousemodel for tauopathies. Treatment with anle138b significantly prolonged the survival of transgenic mice and rescued behavioral deficits as well as synapse and neuron loss in the CA3 region and stratum lucidum of the hippocampus. Our results indicate that reducing tau pathology with the anti-aggregative compound anle138b represents a clinically effective and promising approach for the treatment of human tauopathies. Conclusions: In conclusion, anle138b mitigate tau pathology and may represent a new lead compound for developing a therapy for human tauopathies.