Floyd Sarsoza

Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

BackgroundAmyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.ResultsWe immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.ConclusionSince the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (αAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in αAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric α-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a β barrel structural motif.

A Two-Year Study with Fibrillar β-Amyloid (Aβ) Immunization in Aged Canines: Effects on Cognitive Function and Brain Aβ

Aged canines (dogs) accumulate human-type β-amyloid (Aβ) in diffuse plaques in the brain with parallel declines in cognitive function. We hypothesized that reducing Aβ in a therapeutic treatment study of aged dogs with preexisting Aβ pathology and cognitive deficits would lead to cognitive improvements. To test this hypothesis, we immunized aged beagles (8.4–12.4 years) with fibrillar Aβ1–42 formulated with aluminum salt (Alum) for 2.4 years (25 vaccinations). Cognitive testing during this time revealed no improvement in measures of learning, spatial attention, or spatial memory. After extended treatment (22 vaccinations), we observed maintenance of prefrontal-dependent reversal learning ability. In the brain, levels of soluble and insoluble Aβ1–40 and Aβ1–42 and the extent of diffuse plaque accumulation was significantly decreased in several cortical regions, with preferential reductions in the prefrontal cortex, which is associated with a maintenance of cognition. However, the amount of soluble oligomers remained unchanged. The extent of prefrontal Aβ was correlated with frontal function and serum anti-Aβ antibody titers. Thus, reducing total Aβ may be of limited therapeutic benefit to recovery of cognitive decline in a higher mammalian model of human brain aging and disease. Immunizing animals before extensive Aβ deposition and cognitive decline to prevent oligomeric or fibrillar Aβ formation may have a greater impact on cognition and also more directly evaluate the role of Aβ on cognition in canines. Alternatively, clearing preexisting Aβ from the brain in a treatment study may be more efficacious for cognition if combined with a second intervention that restores neuron health.

Alpha- and beta-secretase activity as a function of age and beta-amyloid in Down syndrome and normal brain.

Aged individuals with Down syndrome (DS) develop Alzheimers disease (AD) neuropathology by the age of 40 years. The purpose of the current study was to measure age-associated changes in APP processing in 36 individuals with DS (5 months-69 years) and in 26 controls (5 months-100 years). Alpha-secretase significantly decreased with age in DS, particularly in cases over the age of 40 years and was stable in controls. The levels of C-terminal fragments of APP reflecting alpha-secretase processing (CTF-alpha) decreased with age in both groups. In both groups, there was significant increase in beta-secretase activity with age. CTF-beta remained constant with age in controls suggesting compensatory increases in turnover/clearance mechanisms. In DS, young individuals had the lowest CTF-beta levels that may reflect rapid conversion of beta-amyloid (Abeta) to soluble pools or efficient CTF-beta clearance mechanisms. Treatments to slow or prevent AD in the general population targeting secretase activity may be more efficacious in adults with DS if combined with approaches that enhance Abeta degradation and clearance.

β-Amyloid deposition and tau phosphorylation in clinically characterized aged cats

The current study describes both Abeta and tau abnormalities that accumulate in the brains of aged (16-21 years), but not young (<4 years) clinically characterized cats. Diffuse plaques that were morphologically different from what is typically observed in the human brain could be detected with 4G8 (Abeta17-24) or an Abeta1-42-specific antibody but not with N-terminal Abeta or an Abeta1-40-specific antibody. SELDI-TOF mass spectrometry experiments indicated that cat brain Abeta consisted almost entirely of Abeta1-42. Markers of tau hyperphosphorylation (AT8 and PHF-1) labeled a subset of neurons in two aged animals. In the hilus of the hippocampus, a subset of AT8 positive neurons showed a sprouting morphology similar to that observed in human brain. Western blot analysis with antibodies against hyperphosphorylated tau indicated that tau is hyperphosphorylated in the aged cat and contains many of the same epitopes found in Alzheimers disease (AD) brain. Thus, the aged cat brain develops AD-related lesions with important morphological and biochemical differences compared to human brain.

Synaptic proteins, neuropathology and cognitive status in the oldest-old

An increasing number of individuals in our population are surviving to over 90 years and a subset is at risk for developing dementia. However, senile plaque and neurofibrillary tangle pathology do not consistently differentiate individuals with and without dementia. Synaptic protein loss is a feature of aging and dementia and may dissociate 90+ individuals with and without dementia. Synaptophysin (SYN), postsynaptic density 95 (PSD-95) and growth-associated protein 43 (GAP-43) were studied in the frontal cortex of an autopsy series of 32 prospectively followed individuals (92-105 years) with a range of cognitive function. SYN protein levels were decreased in individuals with dementia and increased in those with clinical signs of cognitive impairment insufficient for a diagnosis of dementia. SYN but neither PSD-95 nor GAP-43 protein levels were significantly correlated with mini-mental status examination (MMSE) scores. Frontal cortex SYN protein levels may protect neuronal function in oldest-old individuals and reflect compensatory responses that may be involved with maintaining cognition.

Caspase-Cleaved Tau Accumulation in Neurodegenerative Diseases Associated with Tau and α-Synuclein Pathology

Alzheimer’s disease (AD), Pick’s disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB) are diseases associated with the accumulation of tau or α-synuclein. In AD, β-amyloid (Aβ)-associated caspase activation and cleavage of tau at Asp421 (ΔTau) may be an early step in neurofibrillary tangle (NFT) formation. To examine whether ΔTau accumulates in other diseases not characterized by extracellular Aβ accumulation, we examined PiD, PSP, and CBD cases in comparison to those without extensive tau accumulation including frontotemporal lobar degeneration without Pick bodies (FTLD) and control cases. Additionally, we studied ΔTau accumulation in DLB cases associated with intracellular α-synuclein. ΔTau was observed in all disease cases except non-PiD FTLD and controls. These results demonstrate that the accumulation of ΔTau may represent a common pathway associated with abnormal accumulation of intracellular tau or α-synuclein and may be relatively less dependent on the extracellular accumulation of Aβ in non-AD dementias.

Reduced neuronal expression of synaptic transmission modulator HNK‐1/neural cell adhesion molecule as a potential consequence of amyloid β‐mediated oxidative stress: a proteomic approach

Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimers disease (AD). Given that amyloid β (Aβ) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Aβ‐mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two‐dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Aβ‐induced oxidative stress in cultured primary cortical mouse neurons. The Golgi‐resident enzyme glucuronyltransferase (GlcAT‐P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK‐1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Aβ, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK‐1 expression. Moreover, in vivo, HNK‐1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Aβ‐mediated oxidation of GlcAT‐P is impairment of its enzymatic function, thereby disrupting HNK‐1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK‐1 modulates synaptic plasticity and is critically involved in memory consolidation.