Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers
© Kayed et al; licensee BioMed Central Ltd. 2007
Received: 06 August 2007
Accepted: 26 September 2007
Published: 26 September 2007
Amyloid-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.
We 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.
Since 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.
The accumulation of misfolded proteins and peptides as amyloid deposits is a characteristic feature of many degenerative diseases, such as Alzheimer disease (AD), although the pathological significance of these deposits remains unclear. In AD, several different types of deposits containing the amyloid Aβ peptide have been recognized, including dense cored, neuritic, diffuse and "cotton wool" plaques [1–3]. Sites of intracellular accumulation of Aβ have also been identified [4–8]. The relationships and pathological significance of these accumulated deposits remain a matter of debate. The pathological significance of the fibrillar plaques is a matter of debate, since cognitively normal aged individuals frequently have large amounts of fibrillar deposits [9, 10] and soluble oligomeric forms of Aβ correlate better with dementia [11, 12]. It has also been suggested that the large insoluble amyloid deposits may serve as reservoirs that release toxic soluble oligomers . It is widely accepted that diffuse amyloid deposits are "non-fibrillar" based on a lack of binding of fibril-specific dyes, like Congo red (CR) and ThioS. Senile plaques and "cored" plaques stain with CR and ThioS, while most diffuse plaques are negative . Thioflavin dyes have served as the basis for the development of contrast agents to image amyloid accumulation in vivo in humans, although these dyes do not label diffuse plaques in human AD brain nor the amyloid deposits that accumulate in transgenic mouse models of AD.
Plaques have also been characterized immunologically. Monoclonal antibodies specific for the carboxyl terminus of Aβ indicate that diffuse plaques primarily contain Aβn-42, while dense core and neuritic plaques contain both Aβn-40 and Aβn-42 [14, 15]. More recently, conformation-dependent antibodies have been reported that recognize a generic epitope that is specific to many types of amyloid fibrils and not soluble monomer regardless of their sequences [16, 17]. The WO1 antibody has been reported to bind to a generic fibril epitope , but this antibody also recognizes morphologically distinct "protofibrils" . Whether this epitope recognized by WO1 is specific to the fibrillar state or is also displayed on prefibrillar aggregates or oligomers has yet to be determined. Other conformation dependent antibodies (A11) have been reported that specifically recognize a generic epitope common to prefibrillar oligomers and not fibrils, monomers or natively folded precursor proteins . These oligomers are widely believed to represent a primary toxic or pathological species and are called "prefibrillar" because they kinetically precede fibril formation and disappear after fibrils have formed [20, 21]. While A11 stains small focal or punctuate deposits in AD tissue, it does not stain diffuse plaques or other plaque types, indicating that diffuse deposits are not accumulations of prefibrillar oligomers.
Here we report a conformation dependent, fibril specific polyclonal antisera that recognizes a generic epitope that is associated with amyloid fibrils and soluble fibrillar oligomers that is distinct from prefibrillar oligomers. Like other conformation dependent antibodies, it does not recognize natively folded APP or monomer. This antibody may have considerable utility for localizing and quantitating fibril related aggregates in tissues and biological fluids.
Fibril specificity of OC antisera
Characterization of soluble fibrillar oligomers
Conformation dependent immune response is independent of antigen sequence
OC and LOC immunoreactivity in human AD tissue
OC staining of islet amyloid deposits
Amyloidogenic proteins and peptides can adopt a number of distinct assembly states and a key issue is which of these assembly states are more closely associated with pathogenesis. Conformation dependent antibodies that specifically recognize these distinct assembly states offer the potential of providing insight into which distinct assembly states accumulate in disease and are pathologically significant. We have prepared polyclonal antisera that recognize a generic epitope specific to amyloid fibrils. These antibodies are similar to other fibril specific antibodies that have been previously reported [16, 17]. One of the major insights we have gained from examining the properties of OC polyclonal antiserum and comparing it to A11, is that there are at least two distinct types of amyloid oligomers: prefibrillar oligomers and fibrillar oligomers. The idea of fibrillar oligomers may seem counter intuitive because fibrils are often thought of as large insoluble structures, but the fact that OC recognizes small soluble oligomers in the range of dimers to 250 kDa that are not recognized by A11 indicates that these oligomeric species are conformationally related to fibrils and conformationally distinct from prefibrillar oligomers. It is not surprising that small fibrillar oligomers or pieces of fibrils exist, because it is known that fibril assembly is a nucleation-dependent process and these soluble fibrillar oligomers may represent fibril nuclei or seeds.
The size of amyloid oligomers is not a reliable indicator of their conformation or aggregation state. Prefibrillar oligomers and fibrillar oligomers display broadly overlapping size distributions, yet they are conformationally distinct by virtue of the fact that they display mutually exclusive epitopes. Since soluble amyloid oligomers are widely believed to represent the primary pathological species rather than insoluble amyloid fibrils, this raises the question of which type of oligomer is important for pathogenesis and whether prefibrillar and fibrillar oligomers have distinct pathogenic mechanisms. Prefibrillar oligomers have been reported in human AD brain . It is not yet clear whether fibrillar oligomer exist in human AD brain and whether this correlates with pathology, but this is the subject of further studies. Our results also suggest that a lack of staining with thioflavin dyes and Congo red may not be a reliable indicator of non-fibrillar conformation. These dyes do not stain diffuse amyloid deposits although all amyloid deposits including diffuse plaques stain with OC, indicating that they display the fibril-specific epitope. It has been suggested that insoluble amyloid plaques may represent a reservoir for soluble oligomer production. If so, our data would suggest that this would be a source of fibrillar oligomers rather than prefibrillar oligomers, since all types of amyloid plaque deposits stain intensely with OC.
Recent evidence indicates that prefibrillar oligomers are not an obligate intermediate for amyloid fibril formation and that they represent alternative aggregation pathways and in vitro. Fibrils form under conditions that do not support the formation of prefibrillar oligomers and drugs that inhibit prefibrillar oligomer formation have no effect on fibril formation or they actually promote fibril formation [22, 26]. If prefibrillar oligomers are not obligate intermediates for fibril formation in vivo, this would suggest that amyloid fibrils could deposit without forming prefibrillar oligomers. If prefibrillar oligomers are the primary toxic species, the direct formation of fibrils in the absence of prefibrillar oligomers could explain why some cognitively normal individuals have large amounts of insoluble amyloid fibrils.
We also investigated whether the conformation dependent immune response that gives rise to antibodies that recognize generic epitopes associated with distinct aggregations states is independent of the peptide sequence used as an antigen. We immunized rabbits with oligomer mimics and amyloid fibrils derived from the islet amyloid polypeptide and compared the specificity of the immune response to antibodies elicited in response to Aβ derived oligomer mimics and amyloid fibrils. We found that the resulting immune sera are functionally equivalent with the islet oligomer mimic antiserum, I11, recognizing the same prefibrillar oligomers as A11 and the islet fibril antibodies, LOC recognizing the same fibrillar epitope as OC. This suggests that the immune response to amyloid aggregates is predominantly conformation dependent, rather than sequence specific. This observation may have some practical utility. The staining of Aβ amyloid plaques with LOC rules out the possibility that the plaque staining is due to residual Aβ sequence specific immunoreactivity. It also suggests the possibility that oligomers and fibrils may be specifically targeted by vaccination with non-human amyloid sequences that could give rise to a therapeutic immune response while avoiding potential autoimmune complications.
Fibrils and fibrillar amyloid oligomers display a unique generic epitope that is distinct from the generic epitope displayed by prefibrillar oligomers. Conformation dependent antibodies specific for these generic epitopes may have broad utility for specifically targeting these distinct aggregation states in amyloid related degenerative diseases.
Antigen and antibody preparation
Fibril antigens were prepared by stirring 2 mg/ml Aβ42 peptide in 50% HFIP/H2O, 0.02% sodium azide for 7 days. Afterwards, the HFIP was evaporated under a stream of nitrogen and the sample was stirred for an additional 7 days and dialyzed against PBS (molecular weight cut off 10,000 Da). The resulting fibrils were checked by EM and the purity was confirmed by the absence of oligomers using anti-oligomer antibody. The same protocol was used to prepare IAPP fibrils. Aβ and IAPP oligomer mimics were prepared as previously described  The synthesis of IAPP C-terminal thioester analog lacking cysteine residues (KANTATAATQRLANFLVHSSNNFGAILSSTNVGSNTY-SR), was also carried out according to the methods described . The antigens were each used to immunize two New Zealand white rabbits (Pacific Immunology Corp., Ramona, CA, 92065) according to protocols approved by IACUC. Each rabbit immunized with 500 μl of antigen in complete Freund's adjuvant (CFA), and then boosted twice at four week intervals with 500 μl of antigen in Incomplete Freund's Adjuvant (IFA).
Fibril and oligomer preparation
Aβ fibrils and fibrillar oligomers were prepared by dissolving 0.3 mg of lyophilized Aβ42 in 150 ul of hexafluoro-2-propanol (HFIP) for 10–20 minutes at room temperature. The resulting Aβ solution was added to DD H2O in a siliconized Eppendorf tube to 80 uM concentration. After 10–20 min incubation at room temperature, the samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N2 for 10 min to evaporate the HFIP. The sample was then stirred at 500 RPM using a Teflon coated micro stir bar for 24 hours at 22°C. This method was originally reported for preparing A11 positive prefibrillar oligomers , but more recent work indicates that it also produces fibrillar oligomers that are OC positive. Fibrils were separated from fibrillar Aβ42 oligomers by centrifuged at 100,000 × G for 1 hour at 4°C. The supernatant containing fibrillar oligomers and pellet fraction containing fibrils were separated and collected. The pellets were resuspended in an equal volume of H2O. Alternatively, 1 mg of lyophilized Aβ42 was dissolved in 200 μl of DMSO and incubated at room temperature for 10–15 minutes to form fibrillar oligomers. The fibrillar oligomers in DMSO were fractionated according to size using a TSK-GEL SuperSW2000 column (Tosoh Bioscience LLC) in 10 mM Phosphate, pH 7.4 at a flow rate of 0.3 ml/min.
Prefibrillar oligomers that are OC negative were prepared as previously described . Aβ42 stock solutions (2 mM) were prepared by dissolving the lyophilized peptide in 100 mM NaOH followed by water bath sonication for 30 s. The oligomerization reaction was initiated by diluting the stock solution in phosphate buffered saline (PBS), pH = 7.4, 0.02% sodium azide (45 μM final Aβ42 concentration, final pH = 7.4) and incubated at room temperature for up to 15 days. Oligomer formation was monitored by dot blot with A11 and OC polyclonals.
ADDLs were prepared according to Lambert et al . Aβ42 peptide was initially incubated in HFIP at room temperature for 1 hour. The HFIP was evaporated under a gentle stream of N2. The remaining Aβ42 was dissolved in anhydrous DMSO at 5 mM, diluted into cold phenol red-free F12 medium and aged at 4°C for 24 hours. The final peptide concentration was 80 μM.
Q36 fibrils were prepared by dissolving Q36 in 10 mM HEPES/100 mM NaCl at a concentration of 45 μM and incubating with stirring at 25 C for 6 days. Fibril formation was confirmed by EM and ThT. Q36 oligomers were prepared by dissolving Q36 in 1:1 (vol:vol) TFA:HFIP at 2 mM and diluting it to 45 μM in PBS and incubating with stirring for 6 days. Oligomer formation was confirmed with A11 antibody.
ELISA, dot blot, Western blot assays and Immunohistochemistry
ELISA, dot blot and western blots assays were done as previously described . Brain tissue from autopsy cases was provided by the University of California Alzheimer's Disease Research Center and the Institute for Brain Aging & Dementia Brain Tissue Resource. For single-labeling experiments, 50 μm 4% paraformaldehyde-fixed sections containing the hippocampus and entorhinal cortex or the midfrontal cortex from an AD case (Case #1 – 65 year old female, 7.4 hour post mortem interval, MMSE = 21 15 months prior to death with a Braak & Braak stage of VI) were washed with 0.1 M Tris-buffered saline (TBS), pH 7.5, and then pretreated with 3% hydrogen peroxide in 10% methanol to block endogenous peroxidase activity. Sections were subsequently washed in TBS with 0.1% Triton X-100 (TBS-A) and then blocked for thirty minutes in TBS-A with 3% bovine serum albumin (TBS-B). In a series of preliminary studies, the effects of different concentrations of formic acid during a pretreatment phase was determined to have little effect on the extent or intensity of OC labeling of fixed tissue (data not shown) and thus this step was eliminated from the protocol. Sections were incubated overnight at room temperature in OC at a dilution of 1:10,000. Following two washes with TBS-A and a wash in TBS-B, sections were incubated in either goat anti-mouse or goat anti-rabbit biotinylated anti-IgG and then in avidin biotin complex (ABC) (Vector Laboratories, Burlingame, CA, USA). Antibodies were visualized using 3,3'-diaminobenzidine (DAB, Vector Laboratories). For quantification experiments, the frontal cortex from all cases used for quantification was immunostained in single experiments to reduce variability in the image analysis procedures and all immunohistochemical procedures were identical for the OC antibody. To determine the specificity of the OC antibody, IgG purified serum at a concentration of 1:10,000 was preincubated in 10× and 100× concentration of OC peptide for 2 hours at RT. Subsequently, a section from the midfrontal cortex was incubated in the OC antibody/peptide solution and processed for immunohistochemistry. Sections were counterstained with cresyl violet.
Mouse strain and tissue collection
Hemizygous human IAPP transgenic mice (FVB-tg(IAPP)6Jdm/-Avy/A) were characterized in details elsewhere . For this study we used 24 wk old mice. Before collection of mouse pancreata, the heart was perfused with 10 ml of 4% paraformaldehyde. The pancreas was dissected in cold PBS, fat and lymph nodes trimmed, the pancreas weighed, fixed in 4% paraformaldehyde at 4°C for 24 h then frozen in OCT.
Immunofluorescence and confocal microscopy
An additional AD case was used for confocal studies. Case #2 was a 95 year old female with a post mortem interval of 5.5 hours and a MMSE score of 12, 6.3 months prior to death. Sections containing the midfrontal cortex were incubated in OC overnight (1: 5,000) and positive labeling was detected using anti-rabbit IgG Alexa Fluor 568 (Molecular Probes, Eugene, OR; 1:200). Alexa Fluoro 488 (Molecular Probes, Eugene, OR; 1:200) was subsequently used to visualize LOC (1:5,000). The order of the primary antibodies was also reversed in additional experiments and the results were similar. Because both antibodies were raised in the same species (rabbit) an intervening formaldehyde treatment was used between the first and second immunolabel . Sections were allowed to dry on slides prior to rehydration and coverslipping with VectaShield (Vector Laboratories, Burlingame, CA, USA). Confocal images were collected on an Olympus IX70 inverted microscope and a BioRad Radiance 2000 Laser Scanning System using a 40, 60 or 100× objective for image analysis. Each antibody label was excited and scanned using lambda strobing and barrier filters at 510 and 480 nm. A z-series scan at either 0.5 or 1 μm intervals was captured to determine the spatial colocalization characteristics of OC with LOC.
Supported by NIH NS 31230, NIH/NIA ADRC P50 AG16573 and NIH/NIA P01 AG000538, the Cure Alzheimer's Fund and the Larry L. Hillblom Foundation.
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