No description available.

The N-terminally pyroglutamyl-modified β-amyloid (Aβ) peptides are found in abundance in the pathological Alzheimer disease (AD) brain deposits. Such modification not only increases the hydrophobic properties of a given molecule, but also plays a protecting role against proteolytic degradation. This project involved the study of the human type I and type II pyroglutamyl peptidases and their involvement in Aβ processing in AD. Human PcpI has been successfully overexpressed in Escherichia coli strain and purified to homogeneity. The protein displayed significant instability in vitro. To overcome this problem a number of methods were employed such as screening for an optimal protein expression system and buffer composition, site-directed mutagenesis and chemical modification of selected surface residues. This resulted in the selection of the HEPPS buffer system as providing the most stabilising conditions for human PcpI. Improvement in the protein stability enabled initial crystallisation experiments and the identification of favourable conditions for crystal production. Further optimization of this process is needed in order to obtain good quality crystals which are required for structural study. The study on human PcpII involved an extensive screening for optimal expression conditions in bacterial, baculovirus/insect and mammalian systems. The truncated PcpII isoform PcpII/S62-H1024, which lacks the N-terminal transmembrane domain, was successfully expressed and secreted from the HEK 293T cell line using three different pOPIN-based constructs. Moreover, homology modelling of human PcpII catalytic domain was performed, which helped to gain an insight into the three-dimensional structure of the protein and its mode of substrate binding. Lastly, immunohistochemical staining of the human AD brain tissue sections was performed to compare the level and distribution of PcpI and PcpII enzymes between diseased and control cases. The results confirmed that the neurodegenerative conditions lead to the increased synthesis of both enzymes in the cortical AD tissues. Additionally PcpI was shown to be able to participate in the degradation of pGlu-modified Aβ peptides.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Informant‐based assessments of cognition and function are commonly used to differentiate individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) from those who are cognitively normal (CN). However, determining the extent to which informant‐based measures correlate to objective neuropsychological tests is important given the widespread use of neuropsychological tests in making clinical diagnoses of aMCI and AD. The aim of the current study is to determine how well the Alzheimer’s Questionnaire (AQ) correlates with objective neuropsychological tests. Results showed that the AQ correlated strongly with the Mini Mental State Exam (r = ‐0.71) and the Mattis Dementia Rating Scale‐2 (r = ‐0.72), and moderate correlations were noted for the AQ with memory function (Rey Auditory Verbal Learning Test Delayed Recall, r = ‐0.61) and executive function (Trails B, r = 0.53). The AQ also correlated moderately with language function (Boston Naming Test 30‐Item, r = ‐0.44), but showed a weak correlation with visuospatial function (Judgment of Line Orientation, r = ‐0.28). The AQ also correlates particularly well with cognitive screens, showing the strongest correlations with the MMSE (r = ‐0.71) and the DRS‐2 (r = ‐0.72). The findings of this study suggest that the AQ correlates well with several neuropsychological tests, particularly those that assess the domains memory and executive function. These results lend further support to the validity of the AQ as a screening instrument for cognitive impairment as it correlates well with neuropsychological measures used to make clinical diagnoses of aMCI and AD.e sites become involved, thus providing significant feedback for possible course revision.

No description available.

The neuropathological process in Alzheimer's disease (AD) is characterized by both intra and extracellular Aβ42 aggregates. The neuropathological process of AD is complex and the exact cause of Aβ aggregation leading towards neuronal death is yet unknown. Several events are implicated towards the development of AD including changes within the proteome. With more than 30 million people currently affected with AD, there is still no cure for AD. In this project we seek to identify differential protein profiles by undertaking a comparative analysis of the intracellular and extracellular effects of Aβ on the human proteome using two cellular neuronal models: MC65 and SHSY5Y cells, to understand the biochemical pathology underlying AD. We also initiated a compound screening approach which not only identified several small molecules and peptides inhibiting the Aβ cytotoxicity, but also identified several known compounds from the LOPAC library acting as potential inhibitors of intra and extracellular Aβ42 cytotoxicity, thus highlighting the importance of drug repositioning to identify novel compounds in the therapeutic regime of AD which could be categorized as Aβ toxicity inhibitors. A comparative qualitative proteomics approach was undertaken using OFFGEL fractionation. The MS data was analysed through GO, biological pathway and protein interaction analysis using various databases such as UniProtKB, DAVID v6.7, KEGG and String 9.0 for the SHSY5Y cells treated with extracellular Aβ42 and MC65 cells which conditionally express intracellular C99, that is further cleaved to intracellular Aβ. This was followed by validation of 8 proteins by in-cell Western assay (ICW) undertaken using the LI-COR Infrared Imaging System for the cell lysates of control and Aβ42 treated SH-SY5Y as well as Aβ induced MC65 cells. We have also screened a library of 1280 LOPAC compounds on both the cell lines and 9 other compounds previously known as Aβ toxicity inhibitors on MC65 cells. The lead compounds were further characterized using MTT, LDH, ThT and ICW assays. The proteomics methodology undertaken through this project identified several novel proteins specific to intracellular and extracellular Aβ aggregation. The GO, biological pathway analysis and the functional interaction study helped to identify proteins associated from the proteasome pathway to be affected as an effect of Aβ aggregation for both the cells exposed with intra and extracellular Aβ aggregation. The compound screening study also identified several compounds as inhibitors of Aβ cytotoxicity. A-77636, a D1 dopamine receptor agonist was identified as a lead compound to reduce the extracellular Aβ42 cytotoxicity at nM concentration. Moreover, 1,3-Diethyl-8-phenylxanthine and Arecaidine propargyl ester hydrobromide also proved successful in attenuating the extracellular Aβ42 cytotoxicity. Apart from this; SEN1000, SEN304 and Scylloinositol were able to completely attenuate the intracellular Aβ cytotoxicity, whereas two other compounds, 1,3-Dipropyl-8-p-sulfophenylxanthine and 3-Bromo-7-nitroindazole from the LOPAC library proved effective in acting as partial inhibitors of intracellular Aβ aggregation induced cytotoxicity. The ADME profile for most of these compounds is acceptable, therefore these can be considered as therapeutic leads for AD in the future.

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

A large body of research has investigated various aspects of attention in normal aging and Alzheimers disease (AD). Most of the previous studies have shown that divided attention, the ability to attend to two tasks or stimuli simultaneously, declines in both normal aging and AD. In a recent study of attention, Baddeley, Baddeley, Bucks, and Wilcock (2001) reported findings that contrast with other divided attention research. Specifically, they found no effects of aging on divided attention. Taken in combination with their findings of age and AD effects on other aspects of attention, the authors concluded that age-equivalent results on divided attention tasks support the theory that attentional control should be viewed as a fractionated system. Study 1 considered methodological differences between the divided attention tasks used by Baddeley et al., and the tasks used by researchers who have reported age-related differences. Specifically, the effects of task difficulty on age effects were examined. Young, middle-aged, and older adults were compared on a dual-task procedure that combined a secondary visuomotor task (box joining) with a primary verbal task (month reciting) administered at two levels of difficulty. Results showed a significant Age x Task Difficulty interaction. That is, differences among age groups were proportionately greater in the difficult dual-task condition versus the easy condition, suggesting that age-related declines in divided attention may only be detected if tasks are sufficiently difficult.<p> Study 2 examined attention in normal aging and AD. Young adults, older adults, and early-stage AD patients were compared on tasks of selective attention, focal attention, and divided attention, with each task administered at two levels of difficulty. Similar Group x Task Difficulty interaction effects were detected for all attentional tasks, a finding which is more consistent with a general-purpose model than a fractionated model of attention. Study 3 considered attentional tasks from a clinical perspective. Specifically, the attentional tasks utilized in Study 2 were examined with respect to their ability to correctly classify individuals with early-stage AD and normal older adults. Findings showed that all attentional tasks successfully discriminated patients from cognitively healthy older adults, with one task of divided attention showing particularly impressive sensitivity and specificity. Findings of the three studies are discussed with regard to their implications for future research and clinical practice.

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression. / February 2008

A large body of research has investigated various aspects of attention in normal aging and Alzheimers disease (AD). Most of the previous studies have shown that divided attention, the ability to attend to two tasks or stimuli simultaneously, declines in both normal aging and AD. In a recent study of attention, Baddeley, Baddeley, Bucks, and Wilcock (2001) reported findings that contrast with other divided attention research. Specifically, they found no effects of aging on divided attention. Taken in combination with their findings of age and AD effects on other aspects of attention, the authors concluded that age-equivalent results on divided attention tasks support the theory that attentional control should be viewed as a fractionated system. Study 1 considered methodological differences between the divided attention tasks used by Baddeley et al., and the tasks used by researchers who have reported age-related differences. Specifically, the effects of task difficulty on age effects were examined. Young, middle-aged, and older adults were compared on a dual-task procedure that combined a secondary visuomotor task (box joining) with a primary verbal task (month reciting) administered at two levels of difficulty. Results showed a significant Age x Task Difficulty interaction. That is, differences among age groups were proportionately greater in the difficult dual-task condition versus the easy condition, suggesting that age-related declines in divided attention may only be detected if tasks are sufficiently difficult.<p> Study 2 examined attention in normal aging and AD. Young adults, older adults, and early-stage AD patients were compared on tasks of selective attention, focal attention, and divided attention, with each task administered at two levels of difficulty. Similar Group x Task Difficulty interaction effects were detected for all attentional tasks, a finding which is more consistent with a general-purpose model than a fractionated model of attention. Study 3 considered attentional tasks from a clinical perspective. Specifically, the attentional tasks utilized in Study 2 were examined with respect to their ability to correctly classify individuals with early-stage AD and normal older adults. Findings showed that all attentional tasks successfully discriminated patients from cognitively healthy older adults, with one task of divided attention showing particularly impressive sensitivity and specificity. Findings of the three studies are discussed with regard to their implications for future research and clinical practice.