Introduction
The pathological hallmarks of Alzheimer’s disease (AD) are senile plaques and neurofibrillary tangles (NFT) [
1],[
2]. The accumulation of NFT, composed of misfolded, hyperphosphorylated tau proteins [
3], follows a hierarchical spatiotemporal pattern which is consistent with anatomical connections in the brain [
4],[
5]. It therefore appears that NFT deposition spreads from one brain region to the next along major axonal projections, but the underlying mechanism remains unclear. Given the ability of misfolded tau to induce the misfolding of normal tau molecules via a seeding mechanism [
6]-[
10], it has been hypothesized that trans-synaptic transmission of misfolded tau molecules may underlie the spread of tauopathy [
11]-[
13], perhaps analogous to the spread of prion proteins within the brain [
14]. Some have proposed that many neurodegenerative disorders may share the general feature of "prion-like" propagation of misfolded proteins [
15],[
16].
Studies in animal models have demonstrated that tauopathy can spread in the living brain, using either transgenic mice that express mutant human tau proteins specifically in the entorhinal cortex [
11],[
17], or by injecting tau aggregates into specific brain regions [
18],[
19]. However, these processes are relatively inefficient, taking weeks to months to observe, and occur only in situations of high levels of exogenous or transgenic tau. Tau has historically been identified as a microtubule-associated protein localized to the axon of mature neurons [
20],[
21], and a prerequisite for trans-synaptic propagation would be the localization of tau specifically at the synapse. We therefore hypothesized that, for the propagation model to be credible in human diseases, tau would need to be found at the synapse (at least in the disease state); if present at the synapse, the identification of tau species differentially present in pre- or post-synaptic elements, and in AD compared to controls, will test the further hypotheses that misfolded tau accumulates presynaptically before "release" into postsynaptic space, and that tau is mislocalized to the synapse in AD compared to normal neurons.
To test these hypotheses, we isolated and visualized intact, bipartite human neuronal synapses from cortical tissues of control and AD subjects. Using immunofluorescence to detect different forms of tau at bipartite synapses, we found normal tau protein to be symmetrically distributed across presynaptic and postsynaptic terminals in the normal human brain. Misfolded tau in AD-affected brains was also symmetrically distributed on both sides of the synapse, forming sodium dodecyl sulfate (SDS)-resistant oligomers. These data suggest that synaptic tau becomes hyperphosphorylated and misfolded in situ without significant spatial redistribution. Microscopic aggregates of misfolded tau deposited within synapses may represent early signs of neuronal degeneration, agents of synaptic toxicity, and anatomical substrates responsible for the transmission of tauopathy.
Materials and methods
Reagents
Protease inhibitor (cOmplete tablet) was purchased from Roche (Indianapolis, IN). Phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma (St. Louis, MO) and used in 1:1 combination. High-quality Triton X-100 (glass ampule packaging) was purchased from Pierce (Rockford, IL). Mouse monoclonal antibodies DA9 (total tau), PHF1 (pS396/pS404 tau), and Alz-50 (misfolded tau) were gifts of Peter Davies (Albert Einstein College of Medicine). In accordance with original studies of Alz-50 antibody [
22], we found Alz-50 to be weakly reactive against denatured tau in Western blotting after SDS-PAGE. So Alz-50 is a misfolded-conformation-specific tau antibody only under non-denaturing conditions, suitable for immunostaining of fixed cells/tissues.
Rabbit anti-tau (A20024) was purchased from Dako (Glostrup, Denmark); Rabbit anti-PSD95 (#2507) from Cell Signaling (Danvers, MA); Mouse anti-actin (A4700), rabbit anti-actin (A5060), and mouse anti-MAP2 (M4403) from Sigma; Mouse anti-synaptophysin (AB8049) from Abcam (Cambridge, MA); Rabbit anti-histone H3 (05-928) from Millipore (Billerica, MA); Mouse anti-GFAP (MS-1376) from Thermo (Waltham, MA).
Human subjects
Brains from human subjects with a diagnosis of Alzheimer disease or no cognitive deficits were obtained through the Massachusetts Alzheimer’s Disease Research Center and Massachusetts General Hospital Neuropathology Department. All donor tissue was obtained in accord with local and national IRB regulations. Characteristics of control and AD subjects are listed in Table
1.
Table 1
Characteristics of control and AD subjects examined in this study
C1 | 76 | F | Control | NA | 3/3 | 24 | 1 |
C2 | 80 | F | Control | NA | 2/4 | 54 | 1 |
C3 | 76 | M | Control | NA | 3/4 | 48 | 1 |
C4 | 89 | F | Control | NA | 2/3 | 13 | 2 |
C5 | 91 | F | Control | NA | 3/3 | 19 | 2 |
C6 | 71 | M | Control | NA | NA | 5 | 0 |
C7 | 87 | M | Control | NA | NA | 36 | 1 |
A1 | 85 | F | AD | 4 | 3/4 | 10 | 5 |
A2 | 73 | F | AD | 19 | 3/3 | 14 | 5 |
A3 | 84 | F | AD | 16 | 3/4 | 12 | 5 |
A4 | 92 | M | AD | 22 | 4/4 | 12 | 5 |
A5 | 83 | F | AD | 13 | 3/4 | 12 | 5 |
A6 | 82 | M | AD | 6 | 3/4 | 7 | 6 |
A7 | 91 | F | AD | 14 | 3/4 | 9 | 5 |
A8 | 95 | M | AD | NA | 3/3 | 11 | 6 |
Subcellular fractionation and protein extraction
Frozen human cortical tissue was dissected to separate grey matter from white matter, and 200-300 mg of thoroughly thawed grey matter was gently ground in a Potter-Elvehjem homogenizer with 1.5 mL ice-cold buffer A (25 mM HEPES pH 7.5, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2), supplemented with 2 mM DTT, protease inhibitors and phosphatase inhibitors. The homogenate was passed through two layers of 80 μm nylon filters (Millipore) to remove tissue debris, and a 200 μL aliquot was saved. The saved aliquot was mixed with 200 μL of water and 70 μL of 10% SDS, passed through a 27 gauge needle several times to shear DNA, and boiled for 5 min to prepare the total extract, followed by centrifugation at 15,000×g for 10 min to remove insoluble matter.
To prepare filtered synaptoneurosomes, the rest of the homogenate was passed through a 5 μm Supor membrane filter (PALL, Port Washington, NY) to remove large organelles and nuclei, and centrifuged at 1,000×g for 5 min to sediment synaptic terminals. Each pellet was resuspended in buffer A, split into two aliquots, and centrifuged again to yield two synaptoneurosome pellets. Supernatant from the first centrifugation step was clarified by centrifugation at 100,000×g for 1 h to obtain the cytosol fraction. Cytosolic extract was prepared by adding 1.5% SDS and boiling for 5 min. To prepare synaptoneurosome extracts, each synaptoneurosome pellet was mixed with 250 μL buffer B (50 mM Tris pH 7.5, 1.5% SDS, 2 mM DTT) and boiled for 5 min, followed by centrifugation at 15,000×g for 10 min to remove insoluble matter. Synaptoneurosome pellets may be snap frozen in liquid nitrogen and stored at -80°C.
Immunostaining of synaptoneurosomes
Four-well Lab-Tek II CC2 (polyamine pre-coated) chamber slides (Nunc, Rochester, NY) were used for fixing and imaging synaptoneurosomes. Each synaptoneurosome pellet was resuspended in 5 mL of ice-cold buffer C (10 mM HEPES pH 7.9, 0.3 M sucrose) by gentle pipetting. Using a syringe with 27 gauge needle, 200 μL of synaptoneurosome suspension (containing about 25 μg of total protein) was transferred to each chamber well, followed by the addition of 200 μL 2% paraformaldehyde in ice-cold PBS-MC (phosphate buffered saline with 1 mM MgCl2 and 1 mM CaCl2). After 10 min of incubation with 1% paraformaldehyde at 4°C, synaptoneurosomes became fixed and crosslinked to the glass surface. Fixed synaptoneurosomes were washed with PBS-MC (room temperature from this point on) for three times (5 min each), and permeabilized for 10 min with 0.05% Triton X-100 in PBS-MC with 3% bovine serum albumin (BSA, Sigma), followed by three more washes. Slides were blocked with 4% normal goat serum (Invitrogen) and 3% BSA in PBS-MC for 30 min, and then incubated with primary antibodies diluted in PBS-MC with 3% BSA for 90 min, followed by three washes. Secondary antibodies diluted in PBS-MC with 3% BSA were added for 50 min, followed by three washes. The slide was mounted with #1.5 glass coverslip and Prolong Gold Antifade reagent (Invitrogen, Carlsbad, CA).
Primary antibodies for immunostaining included guinea pig anti-VGluT1 (Millipore AB5905, 1:150), chicken anti-MAP2 (Abcam AB5392, 1:100), DA9 (mouse IgG, 1:150), PHF1 (mouse IgG, 1:80), and Alz-50 (mouse IgM, 1:30). Fluorescent secondary donkey antibodies were purchased from Jackson Immunoresearch (West Grove, PA) and used at 1:100 dilutions (anti-guinea pig DyLight 649, anti-chicken Cy3, anti-mouse IgG Alexa 488, and anti-mouse IgM Alexa 488).
Image acquisition and analysis
Immunofluorescence and brightfield images of synaptoneurosomes were acquired on an AxioImager Z1 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x oil immersion objective (N.A. = 1.40). Images were deconvolved with the Iterative Deconvolution plugin (by Bob Dougherty, OptiNav Inc.) in ImageJ software (version 1.44). This 2D deconvolution program required a point spread function (PSF) generated by Diffraction Limit PSF plugin (Bob Dougherty, OptiNav Inc.). For the brightfield image we used a 400 nm PSF; for green fluorescence channel a 509 nm PSF; for red channel a 550 nm PSF; for far red channel a 650 nm PSF. The optimal iteration number was empirically determined to be 12 for brightfield images and 16 for fluorescence images, with the LP filter diameter set at 1.5 pixels. After deconvolution, brightfield and fluorescence images were overlaid in ImageJ (using hyperstacks) and protein colocalization was determined by manual inspection. The cutoff threshold for fluorescence signals (after deconvolution) was generally set as two standard deviations above the mean. For stereological counting, we created a 12×8 grid image which was overlaid with microscope images, and devised a rule to randomly select different grid areas to look for synaptic terminals until the target number was reached. Two-way ANOVA was computed using Graphpad Prism software and t-tests were computed using Excel software.
Classification of synapse morphology
During stereological counting, we defined a synaptic terminal as a brightfield object in the size range of 300-1000 nm with immunofluorescence signals for either presynaptic (VGlut1) or postsynaptic (MAP2) marker. Brightfield objects negative for both synaptic markers were regarded as non-synaptic organelles/vesicles, and these were not quantified, even if they were positively stained by tau antibodies. To qualify as a bipartite synapse, the brightfield object should show a snowman-like structure or a non-spherical shape being elongated or protruded, and pre/post makers needed to be non-overlapping (judged by the centers of mass of the puncta) and overlaid with the brightfield object in a reasonable manner to be considered as adjacent presynaptic and postsynaptic terminals. When synapses were clustered together with other objects under the brightfield, we did not attempt to classify them as bipartite or hemi synapses. There were also tau-positive puncta which did not overlap with brightfield objects and they were excluded from our analysis.
Transmission electron microscopy
Synaptoneurosome pellets were fixed in 2% glutaraldehyde and 2% paraformaldehyde in PBS overnight at 4°C, rinsed, post-fixed in 1% osmium tetroxide, and embedded in LR White resin (Electron Microscopy Sciences, Hatfield, PA) according to manufacturer's protocols. Embedded blocks were cut into 70-nm thin sections on an Ultracut Microtome (Leica, Nussloch, Germany). Images were acquired on a JEOL1011 transmission electron microscope equipped with an ATM digital camera (JEOL USA, Peabody, MA).
Gel electrophoresis and immunoblotting
SDS-denatured protein extracts were subjected to BCA assay (Pierce) to determine protein concentrations. Extracts were boiled again for 3 min after adding 5x sample buffer (250 mM Tris pH 7.5, 5% SDS, 400 mM DTT, 50% glycerol, 0.2% Orange G). Samples were resolved by SDS-PAGE using Bis-Tris 4-12% precast gels (Invitrogen), and transferred to low-fluorescence PVDF or nitrocellulose membranes (Millipore) for immunoblotting, detected using an Odyssey laser scanner (Li-Cor, Lincoln, NE). Blocking buffer and fluorescent secondary antibodies were purchased from Li-Cor and used according to manufacturer's protocols.
Conclusion
In this study we developed a new procedure to image intact synapses isolated from postmortem brain tissues and conducted the first quantitative assessment of synaptic tauopathy in AD. We demonstrated that tau misfolding occurs symmetrically across the synapse with high frequencies of oligomer deposition, revealing for the first time the extraordinary number of synaptic terminals containing microscopic tau inclusions. This methodology would be equally applicable to other neurodegenerative tauopathies such as progressive supranuclear palsy, certain forms of frontotemporal dementia, or corticobasal degeneration [
59]. The underlying reason that AD, but not other tauopathies [
51],[
60], shows a hierarchical pattern of NFT deposition originating in transentorhinal regions still remains unanswered. Comparing the synaptic distribution of tau and analyzing its biochemical state in different tauopathies may provide insights into their respective pathogenesis mechanisms. The discovery of misfolded tau oligomers inside synaptic terminals highlights novel targets for pathogenesis studies and therapeutic interventions. For instance, tau immunotherapy may be able to sequester extracellular misfolded tau after it escapes from pre- or post-synaptic compartments but before it enters another neuron. It remains to be seen if synaptic deposition of misfolded proteins is a common mechanism of neurodegenerative proteinopathies, and imaging of isolated human synapses will be very useful for such investigations.
Competing interests
The authors declare that they have no competing interests.