Absence of amyloid β oligomers at the postsynapse and regulated synaptic Zn²⁺ in cognitively intact aged individuals with Alzheimer’s disease neuropathology

The neuropathological and clinical characteristics of the cases used in this study are summarized in Table 1. The details of the cognitive examination of the study subjects have been reported previously [4, 6, 46] and neuropathological methods are detailed in the Methods section. Groups were designated based on Braak and plaque stages according to CERAD specifications and the Mini Mental State Exam (MMSE) test scores as described in the Methods section. The control group had Braak stage 0–2 and no more than sparse neuritic plaques and were cognitively intact on annual neuropsychiatric testing, as summarized by terminal MMSE scores >25. The AD group had Braak scores of 6 and moderate to frequent neuritic plaques with below normal MMSE scores (average of 9). The NDAN cases had Braak scores that ranged from 4 to 6 and a range of neuritic plaque densities, but typically with moderate densities of neuritic plaques. The MMSE scores in this group were comparable to those in age-matched normal individuals (>25) with an average of 28. The postmortem interval (PMI) was <24 h, except for two cases. Tissue morphology was well preserved in all samples and there was no sign of protein degradation in Western blots performed on protein extracts from samples used in this study.

The pathological analysis described above for AD and NDAN cases confirmed that these two groups have a comparable presence of Aβ plaques and NFTs, further corroborating the notion that cognitive diversity between AD and NDAN individuals could not be ascribed to different extents of these two AD-related neuropathological features [5]. Figure 1 shows representative panels of the immunohistochemical detection of Aβ and phosphorylated tau (Figure 1A) and Bielschowsky staining of amyloid plaques and NFTs (Figure 1B) in the hippocampus from representative cases used in the present study. Furthermore, the levels of Aβ_1-42 were equally increased in NDAN and AD as determined by a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (Figure 1C).

In addition to soluble monomers, Aβ_1-42 is normally present in the affected human brain in structurally distinct aggregated forms, including large fibrils, which are the main component of senile plaques and small soluble oligomers, which are believed to be the most neurotoxic Aβ species [47‐49], We therefore first determined which of these low molecular weight (LMW) Aβ oligomeric species were present in the hippocampus of NDAN cases as compared to AD cases. Western blot analysis revealed that LMW Aβ species of ~4, 8, and 12 kDa were detectable in soluble fractions from hippocampi of both AD and NDAN cases (Figure 1D). Statistical analysis applied to densitometry values obtained following this approach showed that the levels of LMW Aβ species were significantly increased in both groups compared to control (Figure 1E). Thus, the presence of LMW Aβ species in the NDAN samples eliminates the possibility that these individuals remain cognitively intact due to lack of highly neurotoxic Aβ oligomers.

These data highlight the presence of comparable levels of plaques, tangles, Aβ _1–42 levels, and LMW Aβ oligomers between AD and NDAN.

Aβ oligomers have been shown to accumulate at the synapses where they induce dysfunctional changes that impair synaptic integrity [18, 20, 49]. Therefore, to further assess the localization of the LMW Aβ species detected in the AD and NDAN brains, we performed a synaptic fractionation that separates presynaptic and postsynaptic fractions [50] and probed the postsynaptic fractions for the presence of Aβ by Western blotting. Successful synaptic fractionation of the brain tissues was confirmed by probing the Western blots of the different fractions with antibodies directed against pre- and postsynaptic markers as illustrated in Figure 2A. The fractionation was successful for each group (age-matched control, AD, and NDAN) as shown by the enrichment of PSD95 in the PSD fraction (Figure 2B).

When blots were probed for Aβ, we found that LMW Aβ species were highly concentrated in the postsynaptic fractions from AD hippocampi, but completely absent from aged-matched controls and NDAN cases (Figure 2C). Densitometric analysis (Figure 2D) confirmed that LMW Aβ species were abundant at the PSD isolated from AD hippocampi but absent (undetectable above background) in NDAN cases. This corroborates previous reports that Aβ selectively targets the PSD in AD [17, 51].

To confirm that the LMW Aβ species found at the PSD from AD hippocampi were oligomers, an Aβ oligomer-specific antibody, NU4, was utilized [52]. The representative Western blot in Figure 2E demonstrates that the low molecular Aβ species detected in AD samples are indeed oligomers (particularly dimers and trimers, as assessed by comparison to synthetic (Synt) Aβ oligomers). On the other hand, neither dimeric nor trimeric Aβ species were detectable in the NDAN samples, and this was statistically confirmed by densitometric analysis of the blots (Figure 2F). Interestingly, an Aβ tetrameric species was present in the sample from all groups, including the control samples, which suggests that this species is not uniquely associated with clinically manifested AD. These data demonstrate that while present in the soluble fractions from both AD and NDAN cases, Aβ oligomers are highly associated with the PSD only in AD hippocampi, but are absent in the PSD prepared from NDAN cases.

Our previous studies have shown that one downstream effect of Aβ oligomers dysfunctionally impacting synapses is the decrease of active, phosphorylated CREB (pCREB), a transcription factor essential for synaptic plasticity and memory function [12, 16, 53]. The cognitive integrity of NDAN individuals suggests that synaptic function is preserved in these individuals, which would thus be consistent with the observed absence of Aβ at the postsynapse. We therefore determined the expression of neuronal pCREB in each of the samples using immunohistochemistry coupled to confocal microscopy (Figure 3A) as an index of functional synaptic integrity. When pCREB levels were quantified in granular neurons of the dentate gyrus (DG) and in pyramidal neurons of the cornu ammonis region 3 (CA3) in postmortem human hippocampus (Figure 3B) we found significantly fewer pCREB positive neurons in both cell populations in subjects with AD. Conversely, despite the presence of abundant Aβ and NFTs in the NDAN hippocampi, levels of pCREB were similar to those of control cases. Levels of pCREB were also measured by Western blot in total homogenate fractions from each group (Additional File 1). Consistent with the immunohistochemistry results, we found that there was a decrease in the AD samples, but not NDAN, which however did not reach statistical significance. This result is not completely unexpected if one considers that total homogenate does contain proteins contributed from both neurons and glial cells, both of which express CREB, thus reducing the sensitivity of Western blotting to detect changes in pCREB levels occurring solely in neurons (as our immunohistochemistry results seem to indicate). Notwithstanding this technical limitation, direct observation by immunohistochemistry and Western blot detection collectively indicate that pCREB levels in hippocampal neurons are not reduced in NDAN as compared to AD cases, thus suggesting that synaptic integrity in NDAN neurons is indeed preserved.

The exclusion of Aβ at the postsynapses in NDAN individuals suggests that mechanisms involved in the pathological targeting of Aβ to synapses may be altered or absent in these individuals. Among these, synaptic Zn²⁺ can coordinate with Aβ oligomers and target them to the synapse where the oligomers can induce toxic effects [26]. We therefore measured Zn²⁺ levels to determine if alterations in Zn²⁺ concentration are associated with the absence of Aβ oligomers at the postsynapse in NDAN individuals. Soluble fractions from hippocampi were processed as described in the methods and Zn²⁺ levels were analyzed using GF-AAS. Our results show that the AD specimens had significantly higher Zn²⁺ concentrations than control (Figure 4A). Zn²⁺ levels in the NDAN tissue were also higher than control, but significantly lower than those observed in AD samples.

Zn²⁺ in the soluble fractions would include both protein-bound and releasable free Zn²⁺. The releasable Zn²⁺ stored into presynaptic vesicles and released during synaptic activity becomes available to coordinate with proteins present at the synapse [54, 55], including Aβ oligomers. Therefore, to achieve a better assessment of the levels of the releasable Zn²⁺ pool, synaptic vesicles were isolated for analysis of Zn²⁺ content (Figure 4B). The levels of Zn²⁺ in the synaptic vesicles were increased in both AD and NDAN samples as compared to brain samples from age-matched control individuals.

The ZnT3 transports Zn²⁺ into synaptic vesicles and is transcriptionally regulated by Zn²⁺[54]. Previously, mRNA and protein expression of this transporter was found to be decreased in AD brain [30, 32]. Immunoblots probing for ZnT3 confirmed this finding and also showed that ZnT3 expression in NDAN was maintained at control levels (Figure 4C). Taken together these data suggest that despite comparable levels of vesicular Zn²⁺, but higher expression of the ZnT3 and lower Zn²⁺ in the soluble fraction, the regulation of Zn²⁺ homeostasis in NDAN individuals may be better preserved in comparison to AD cases, thus possibly reducing the Zn²⁺: Aβ interaction and subsequent Aβ targeting to the PSD.

Frozen mid-hippocampus tissue was obtained from the Oregon Brain Bank at Oregon Health and Science University (OHSU) in Portland, OR (See Table 1). Donor subjects were enrolled and clinically evaluated in studies at the NIH-sponsored Layton Aging and AD Center (ADC) at OHSU. Subjects were participants in brain aging studies at the ADC and received annual neurological and neuropsychological evaluations, with a clinical dementia rating (CDR) assigned by an experienced clinician. Controls had normal cognitive and functional examinations. The AD subjects were diagnosed by a clinical team consensus conference, met the National Institute for Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorder Association diagnostic criteria for clinical AD, had a CDR of greater than 1.0, and neuropathologic confirmation at autopsy (after informed consent). Tissue use conformed to institutional review board-approved protocols. Neuropathologic assessment conformed to National Institute on Aging-Reagan consensus criteria. All brain tissue was examined by a neuropathologist for neurodegenerative pathology including neurofibrillary tangles and neuritic plaques. Using standardized CERAD criteria [60], cases were assigned an amyloid score based on the deposition of amyloid plaques in the brain (0 = no plaques, 1 = sparse plaques, 2 = moderate plaques, and 3 = dense plaques), and a Braak stage (0–6; with 6 being the most severe) indicative of the level and location of hyper-phosphorylated tau tangles [3]. In addition to the pathological information detailed above, demographical data were received along with the frozen tissue. These included age, sex, and MMSE score [61] for each case.

To ensure that the variations in postmortem interval (PMI) did not affect the measurements, a correlation analysis between PMI values and results obtained in the various assays presented here was performed using a Pearson’s correlation test. No correlation was found for any of the experiments presented (Additional File 2), and therefore observed differences could not be attributed to differences in non-specific postmortem tissue degradation among the various groups.

Five-mm sections of the mid hippocampus were brought out of storage at −80°C and equilibrated to −20°C before embedding in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrence, CA, USA). Ten μm sections were cut and affixed to Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA), for further storage as needed at −80°C. After equilibrating to room temperature, sections were rinsed in 0.1 M PBS and then fixed in ice-cold 4% paraformaldehyde for 15 min. Sections were then washed in 0.1 M PBS, followed by blocking and permeabilization for 1 h in 0.1 M PBS containing 10% goat serum, 0.03% Triton-X, and 0.1% phosphatase inhibitor (Thermo Fisher Scientific). Incubation with the primary antibodies, 4 G8 (for Aβ from Covance, Princeton Township, NJ, USA), phosphorylated tau (Ser 202, Pierce Biotechnology, Rockford, IL, USA) CREB, phosphorylated CREB (Ser 133) and NeuN (Millipore, Billerica, MA, USA), in 0.1 M PBS containing 10% serum and 0.1% phosphatase inhibitor was carried out overnight at room temperature. Following washing, slides were incubated for 1 h with Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA, USA) in 0.1 M PBS containing 10% serum and 0.1% phosphatase inhibitor. Slides were rinsed twice in PBS and once in distilled water before a 10 min incubation with 0.3% Sudan Black B (EMD Chemicals, Gibbstown, NJ, USA) in 70% ethanol to block lipofuscin autofluorescence [62]. After rinsing in distilled deionized water, Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) was applied (Vector Laboratories, Burlingame, CA, USA), and coverslips were mounted and nail polish was used to seal the edges.

High-resolution images (1024x1024 TIFF) were acquired using a confocal laser-scanning module (Bio-Rad Radiance 2000 with LaserSharp software, Hercules, CA, USA) mounted on a Nikon E800 upright microscope. For these analyses the 20x//0.75NA, 40x/0.95NA and 60x-oil/1.4NA objectives were used (Nikon, Melville, NY, USA) Images were acquired with a blue diode and krypton lasers of 488 nm and 568 nm excitation. Images for comparisons were acquired with constant settings for laser power, detector gain, amplification gain, and offset.

The modified Bielschowsky stain was performed as described previously as a component of the neuropathological assessment [60].

The soluble fraction used for this assay was generated using 200–300 mg of mid-hippocampus. The tissue was homogenized using a 1 ml syringe with a 20 gauge needle in 50 mM Tris–HCl buffer (pH = 7.6) with 0.01% NP-40, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The homogenate was centrifuged for 5 min at 3000 rpm at 4°C and the supernatant collected for analysis [10]. Samples were normalized for protein content and diluted 1:100 in Standard Diluent Buffer provided in the ELISA kit (Invitrogen). The ELISA was performed according to the manufacturer’s directions. All samples were measured in duplicate at 450 nm.

To prepare oligomeric Aβ, lyophilized Aβ aliquots (0.3 mg) were dissolved in 0.2 ml 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and then added to 0.7 ml H₂O in Eppendorf tubes. Tubes were loosely capped and samples stirred on a magnetic stirrer under a fume hood for 48 h and then used within 36 h. Quality of Aβ preparations was routinely checked by Western blot employing both NU4 and 6E10 antibodies.

Primary antibodies used were synaptophysin (Millipore), PSD95 (Cell Signaling, Danvers, MA, USA), and ZnT3 (LifeSpan Biosciences, Seattle, WA, USA) The monoclonal antibody 6e10 from Covance was used to detect Aβ. The Aβ oligomer-specific antibody, NU4 (a gift from the laboratory of William L. Klein), was used to detect LMW oligomers. Ponceau S staining of the membranes was performed to ensure equal protein loading before incubation with antibodies. In addition, tubulin or GAPDH (Cell Signaling) was used as a loading control. After incubation with appropriate HRP-conjugated (Sigma-Aldrich) or fluorescent secondary antibody (1:10,000) (LI-COR Biosciences, Lincoln, NE, USA), the membrane was either imaged after ECL incubation or scanned directly by an Odyssey infrared fluorescent imaging system (LI-COR Biosciences). Band densities were analyzed using Quantity One (Bio-Rad) or Odyssey software and normalized using the measured loading control densities. Each group was represented on each blot. Differences between groups were determined using a one-way ANOVA followed by Tukey post-hoc analysis.

Different loading controls were utilized depending on the sample fraction. This approach was necessary because many housekeeping proteins such as tubulin and GAPDH have altered expression and localization in aging and AD [64, 65]. A variety of housekeeping proteins and total protein stains [66] were tested in preliminary experiments to determine the most appropriate loading control for each fraction. The loading control whose levels did not change depending on disease state was used for that particular protein fraction.

All laboratory processing was performed under clean contaminant free conditions, to minimize external metal contamination. Each sample was evaporated to dryness in a drying oven at 80 ⁰ C. Digestion of the residue was carried out with a well established procedure [67] using 0.5 mL of 30% hydrogen peroxide (GFS Chemicals, Powell, OH, USA) at 70 ⁰ C for 18–24 h followed by 0.10 mL of Ultra-pure nitric acid (GFS Chemicals) until completely ashed. The digested white ash was dissolved in 4.0 mL of Milli-Q deionized distilled water. Each digested sample was further suitably diluted 1:20, 1:40, 1:50, 1:80, 1:100 or 1:200 (v/v) using Milli-Q deionized distilled water prior to analysis.

Concentrations of Zn²⁺ in the diluted digested samples were determined by GF-AAS as described previously [63]. A Varian Instruments Model-240Z Zeeman atomic absorption spectrophotometer (Varian, Inc., Walnut Creek, CA, USA) equipped with a Varian GTA-120 graphite tube analyzer, a PSD-120 programmable sample dispenser, a Varian UltrAA high intensity boosted hollow cathode lamp were routinely used to measure Zn²⁺ at low parts per billion (ppb) levels in solution. Pyrolytically coated Varian graphite partition tubes were used for all GF-AAS analyses. Argon gas (0.3 L/min flow) was used to protect and purge the graphite tubes during the furnace program steps, and the data acquisitions were carried out using Varian SpectrAA software.

The auto-sampler cups were washed initially with 0.5 N Ultra-pure nitric acid followed by Milli-Q deionized distilled water prior to use. Calibration standards (0.0, 0.5, 1.0, 1.5, and 2.0 micrograms per liter (ppb)) of Zn²⁺ were prepared in Milli-Q deionized distilled water and run with each set of samples and the absorbance was measured at 213.9 nm wavelength. Precision and accuracy of the method were routinely checked by digesting known weights of SRM1577a standard liver powder (NIST, Bethesda, MD, USA) and analyzing them routinely along with these samples. These liver powders contain known certified amounts of several metals including Zn²⁺.

For immunohistochemistry, analysis was performed with ImageJ (NIH) and a set of plug-ins developed by Wright Cell Imaging Facility (http://​www.​uhnres.​utoronto.​ca/​facilities/​wcif/​imagej/​). Two images per region (DG and CA3) were analyzed for each clinical case. A blind counter quantified the number of neurons in the field of view, and how many of these exhibited robust nuclear pCREB immunoreactivity. This value is expressed as the percentage of neurons that are pCREB positive. Microsoft Excel was used for graph production and StatPlus or Sigmaplot were used for data analysis. One-way ANOVA with a Fisher post hoc test was used to determine statistical significance. If the data failed the normality test (Shapiro-Wilk), a Bonferroni correction was made before statistical analysis. Data represents mean ± SEM.