Thomas Dyrks

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimers disease‐specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy‐terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C‐terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor‐product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post‐translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Copper-Dependent Inhibition of Human Cytochrome c Oxidase by a Dimeric Conformer of Amyloid-β1-42

In studies of Alzheimers disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-β (Aβ) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Aβ peptide (Aβ1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Aβ1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific “aging” of the Aβ1-42 solution. Maximal COX inhibition occurred when using Aβ1-42 solutions aged for 3-6 h at 30°C. The level of Aβ1-42-mediated COX inhibition increased with aging time up to ∼6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Aβ as the only Aβ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimers model mouse (Tg2576) revealed abundant Aβ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Aβ is associated with brain mitochondria and that Aβ1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Aβ-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimers disease.

Curcumin-Derived Pyrazoles and Isoxazoles: Swiss Army Knives or Blunt Tools for Alzheimer's Disease?

Curcumin binds to the amyloid β peptide (Aβ) and inhibits or modulates amyloid precursor protein (APP) metabolism. Therefore, curcumin‐derived isoxazoles and pyrazoles were synthesized to minimize the metal chelation properties of curcumin. The decreased rotational freedom and absence of stereoisomers was predicted to enhance affinity toward Aβ42 aggregates. Accordingly, replacement of the 1,3‐dicarbonyl moiety with isosteric heterocycles turned curcumin analogue isoxazoles and pyrazoles into potent ligands of fibrillar Aβ42 aggregates. Additionally, several compounds are potent inhibitors of tau protein aggregation and depolymerized tau protein aggregates at low micromolar concentrations.

Induced LC degeneration in APP/PS1 transgenic mice accelerates early cerebral amyloidosis and cognitive deficits.

Degeneration of locus ceruleus neurons and subsequent reduction of norepinephrine concentration in locus ceruleus projection areas represent an early pathological indicator of Alzheimers disease. In order to model the pathology of the human disease and to study the effects of norepinephrine-depletion on amyloid precursor protein processing, behaviour, and neuroinflammation, locus ceruleus degeneration was induced in mice coexpressing the swedish mutant of the amyloid precursor protein and the presenilin 1 DeltaExon 9 mutant (APP/PS1) using the neurotoxin N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (dsp4) starting treatment at 3 months of age. Norepinephrine transporter immunolabelling demonstrated severe loss of locus ceruleus neurons and loss of cortical norepinephrine transporter starting as early as 4.5 months of age and aggravating over time. Of note, dsp4-treated transgenic mice showed elevated amyloid beta levels and impaired spatial memory performance at 6.5 months of age compared to control-treated APP/PS1 transgenic mice, indicating an accelerating effect on cerebral amyloidosis and cognitive deficits. Likewise, norepinephrine-depletion increased neuroinflammation compared to transgenic controls as verified by macrophage inflammatory protein-1alpha and -1beta gene expression analysis. Exploratory activity and memory retention was compromised by age in APP/PS1 transgenic mice and further aggravated by induced noradrenergic deficiency. In contrast, novel object recognition was not influenced by norepinephrine deficiency, but by the APP/PS1 transgene at 12 months. Overall, our data indicate that early loss of noradrenergic innervation promotes amyloid deposition and modulates the activation state of inflammatory cells. This in turn could have had impact on the acceleration of cognitive deficits observed over time.

Radiation Dosimetry of β-Amyloid Tracers 11C-PiB and 18F-BAY94-9172

β-Amyloid (Aβ) imaging has great potential to aid in the diagnosis of Alzheimer disease and the development of therapeutics. The radiation dosimetry of Aβ radioligands may influence their application; therefore, we calculated and compared the effective doses (EDs) of 11C-PiB and a new 18F-labeled ligand, 18F-BAY94-9172. Methods: Attenuation-corrected whole-body scans were performed at 0, 15, 30, 45, and 60 min after injection of 350 ± 28 MBq (mean ± SD) of 11C-PiB in 6 subjects and at 0, 20, 60, 120, and 180 min after injection of 319 ± 27 MBq of 18F-BAY94-9172 in 3 subjects. Coregistered CT was used to define volumes of interest (VOIs) on the PET images. The source organs were the brain, lungs, liver, kidneys, spleen, and vertebrae. The VOIs for the contents of the gallbladder, urinary bladder, lower large intestine, upper large intestine, and small intestine were also defined. Total activity in each organ at each time point was calculated by use of reference organ volumes. The resultant time–activity curves were fitted with constrained exponential fits, and cumulated activities were determined. A dynamic bladder voiding model was used. The OLINDA/EXM program was used to calculate the whole-body EDs from the acquired data. Results: For 11C-PiB, the highest absorbed doses were in the gallbladder wall (44.80 ± 29.30 μGy/MBq), urinary bladder wall (26.30 ± 8.50 μGy/MBq), liver (19.88 ± 3.58 μGy/MBq), and kidneys (12.92 ± 3.37 μGy/MBq). The ED was 5.29 ± 0.66 μSv/MBq. For 18F-BAY94-9172, the highest doses were also in the gallbladder wall (132.40 ± 43.40 μGy/MBq), urinary bladder wall (24.77 ± 7.36 μGy/MBq), and liver (39.07 ± 8.31 μGy/MBq). The ED was 14.67 ± 1.39 μSv/MBq. Conclusion: The estimated organ doses for 11C-PiB were comparable to those reported in earlier research. With the doses used in published studies (300–700 MBq), the EDs would range from 1.6 to 3.7 mSv. The ED of 18F-BAY94-9172 was 30% lower than that of 18F-FDG and, at the published dose of 300 MBq, would yield an ED of 4.4 mSv. The dosimetry of both Aβ radioligands is suitable for clinical and research applications.

A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system

The binding of two radiolabelled analogues (N-(5-[125I]Iodo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)acetamide ([125I]desfluoro-DAA1106) and N-(5-[125I]Fluoro-2-phenoxyphenyl)-N-(2-[125I]Iodo-5-methoxybenzyl)acetamide ([125I]desmethoxy-DAA1106) of the peripheral benzodiazepine receptor (PBR) (or TSPO, 18kDa translocator protein) ligand DAA1106 was examined by in vitro autoradiography on human post mortem whole hemisphere brain slices obtained from Alzheimers disease (AD) patients and age-matched controls. Both [(125)I]desfluoro-IDAA1106 and [(125)I]desmethoxy-IDAA1106 were effectively binding to various brain structures. The binding could be blocked by the unlabelled ligand as well as by other PBR specific ligands. With both radiolabelled compounds, the binding showed regional inhomogeneity and the specific binding values proved to be the highest in the hippocampus, temporal and parietal cortex, the basal ganglia and thalamus in the AD brains. Compared with age-matched control brains, specific binding in several brain structures (temporal and parietal lobes, thalamus and white matter) in Alzheimer brains was significantly higher, indicating that the radioligands can effectively label-activated microglia and the up-regulated PBR/TSPO system in AD. Complementary immunohistochemical studies demonstrated reactive microglia activation in the AD brain tissue and indicated that increased ligand binding coincides with increased regional microglia activation due to neuroinflammation. These investigations yield further support to the PBR/TSPO binding capacity of DAA1106 in human brain tissue, demonstrate the effective usefulness of its radio-iodinated analogues as imaging biomarkers in post mortem human studies, and indicate that its radiolabelled analogues, labelled with short half-time bioisotopes, can serve as prospective in vivo imaging biomarkers of activated microglia and the up-regulated PBR/TSPO system in the human brain.

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimers disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[(18)F]FMeNER-D(2). (S,S)-[(18)F]FMeNER-D(2) appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[(18)F]FMeNER-D(2) in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[(18)F]FMeNER-D(2) as a molecular imaging biomarker in AD.

MOLECULAR PATHOLOGY OF THE AMYLOID A4 PRECURSOR OF ALZHEIMER??S DISEASE

The precursor of the Alzheimers disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4. We suggest that besides proteolytic cleavage, other events, such as membrane damage are primary events that precede the release of the small, aggregating amyloid A4 subunit.

O1-04-02: Initial results from human studies of a novel F-18 PET ligand for brain beta-amyloid imaging

with best practice methods to deliver care primarily via small group homes and that dementia care takes up a disproportionate amount of staff time in small care settings. Conclusions: To address early stage dementia related services in the most effective manner, a concerted effort needs to be in place to aid local service entities adapt services to dementia-related presentations among ID clientele, set up coordinated training for staff, secure funds for adapting group homes for community “dementia-capable” care, and construction of clinical support services and augmentation of family support services for parents and other kin carers.