Faculty Bibliography

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms - all known pathogenic features of these protein folding disorders - suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.

AIMS/OBJECTIVE:The deposition of amyloid-β (Aβ) peptides in the form of extracellular plaques in the brain represents one of the classical hallmarks of Alzheimer's disease (AD). In addition to "full length" Aβ starting with aspartic acid (Asp-1), considerable amounts of various shorter, N-terminally truncated Aβ peptides have been identified by mass spectrometry in autopsy samples from individuals with AD. METHODS:Selectivity of several antibodies detecting full-length, total or N-terminally truncated Aβ species has been characterized with capillary isoelectric focusing assays using a set of synthetic Aβ peptides comprising different N-termini. We further assessed the N-terminal heterogeneity of extracellular and vascular Aβ peptide deposits in the human brain by performing immunohistochemical analyses using sporadic AD cases with antibodies targeting different N-terminal residues, including the biosimilar antibodies Bapineuzumab and Crenezumab. RESULTS:showed a much weaker staining of extracellular plaques and tended to accentuate cerebrovascular amyloid deposits, antibodies detecting Aβ starting with phenylalanine at position 4 of the Aβ sequence showed abundant amyloid plaque immunoreactivity in the brain parenchyma. The biosimilar antibody Bapineuzumab recognized Aβ starting at Asp-1 and demonstrated abundant immunoreactivity in AD brains. DISCUSSION/CONCLUSIONS:specific antibodies, Bapineuzumab displayed stronger immunoreactivity on fixed tissue samples than with SDS-denatured samples on Western blots. This suggests conformational preferences of this antibody. The diverse composition of plaques and vascular deposits stresses the importance of understanding the roles of various Aβ variants during disease development and progression in order to generate appropriate target-developed therapies.

Impaired clearance in the Alzheimer's Disease (AD) brain is key in the formation of Aβ parenchymal plaques and cerebrovascular deposits known as cerebral amyloid angiopathy (CAA), present in >80% of AD patients and ~50% of non-AD elderly subjects. Aβ deposits are highly heterogeneous, containing multiple fragments mostly derived from catabolism of Aβ40/Aβ42, which exhibit dissimilar aggregation properties. Remarkably, the role of these physiologically relevant Aβ species in cerebrovascular injury and their impact in vascular pathology is unknown. We sought to understand how heterogeneous Aβ species affect cerebral endothelial health and assess whether their diverse effects are associated with the peptides aggregation propensities. We analyzed cerebral microvascular endothelial cell (CMEC) viability, blood-brain barrier (BBB) permeability, and angiogenesis, all relevant aspects of brain microvascular dysfunction. We found that Aβ peptides and fragments exerted differential effects on cerebrovascular pathology. Peptides forming mostly oligomeric structures induced CMEC apoptosis, whereas fibrillar aggregates increased BBB permeability without apoptotic effects. Interestingly, all Aβ species tested inhibited angiogenesis in vitro. These data link the biological effects of the heterogeneous Aβ peptides to their primary structure and aggregation, strongly suggesting that the composition of amyloid deposits influences clinical aspects of the AD vascular pathology. As the presence of predominant oligomeric structures in proximity of the vessel walls may lead to CMEC death and induction of microhemorrhages, fibrillar amyloid is likely responsible for increased BBB permeability and associated neurovascular dysfunction. These results have the potential to unveil more specific therapeutic targets and clarify the multifactorial nature of AD.

Aβ deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aβ_1-40 and Aβ_1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aβ variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aβ_4-42 exhibits a high tendency to form β-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aβ_4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aβ_4-42. An Aβ variant with a double truncation at phenylalanine-4 and leucine 34, (Aβ_4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aβ species in AD pathophysiology.

After the publication of this article [1], we became aware that there were errors in Figs. 4 and 31.

BACKGROUND:Mounting evidence points to a crucial role of amyloid-β (Aβ) in the pathophysiology of Alzheimer's disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not surprising that mitochondrial dysfunction is tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to Aβ, still remain elusive. METHODS:We have used classic biochemical and immunocytochemical approaches together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligAβ in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules. RESULTS:Our findings indicate that oligomeric Aβ induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated Aβ-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of Aβ-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested identified the transcription factor Nrf2-compromised by age and downregulated in AD and transgenic models-as their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their Aβ protective action. CONCLUSIONS:Our study provides insights into the complex molecular mechanisms triggered by oligAβ which profoundly affect mitochondrial performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies.

Extensive parenchymal and vascular Abeta deposits are pathological hallmarks of Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Abeta heterogeneity likely resulting from the action of multiple proteolytic enzymes. In spite of the numerous studies focusing in Abeta, the relevance of N- and C-terminal truncated species for AD pathogenesis remains largely understudied. In the present work, using novel antibodies specifically recognizing Abeta species N-terminally truncated at position 4 or C-terminally truncated at position 34, we provide a clear assessment of the differential topographic localization of these species in AD brains and transgenic models. Based on their distinct solubility, brain N- and C-terminal truncated species were extracted by differential fractionation and identified via immunoprecipitation coupled to mass spectrometry analysis. Biochemical/biophysical studies with synthetic homologues further confirmed the different solubility properties and contrasting fibrillogenic characteristics of the truncated species composing the brain Abeta peptidome. Abeta C-terminal degradation leads to the production of more soluble fragments likely to be more easily eliminated from the brain. On the contrary, N-terminal truncation at position 4 favors the formation of poorly soluble, aggregation prone peptides with high amyloidogenic propensity and the potential to exacerbate the fibrillar deposits, self-perpetuating the amyloidogenic loop. Detailed assessment of the molecular diversity of Abeta species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Abeta truncations to the disease pathogenesis and their potential as novel therapeutic targets.

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aβ heterogeneity likely resulting from the action of multiple proteolytic enzymes. This chapter describes a sequential extraction protocol allowing the differential fractionation of soluble and deposited Aβ species taking advantage of their differential solubility properties. Soluble Aβ is extracted by water-based buffers like phosphate-buffered saline-PBS-whereas pre-fibrillar and fibrillar deposits, usually poorly soluble in PBS, are extractable in detergent containing solutions or more stringent conditions as formic acid. The extraction procedure is followed by the biochemical identification of the extracted Aβ species using Western blot and a targeted proteomic analysis which combines immunoprecipitation with MALDI-ToF mass spectrometry. This approach revealed the presence of numerous C- and N-terminal truncated Aβ species in addition to Aβ1-40/42. Notably, the more soluble C-terminal cleaved fragments constitute a main part of PBS homogenates. On the contrary, N-terminal truncated species typically require more stringent conditions for the extraction in agreement with their lower solubility and enhanced aggregability. Detailed assessment of the molecular diversity of Aβ species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Aβ truncations to AD pathogenesis and their potential as novel therapeutic targets.