Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits | PNAS Research Article Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits Megan E. Larson, Susan J. Greimel, Fatou Amar, Michael LaCroix, Gabriel Boyle, Mathew A. Sherman, Hallie Schley, Camille Miel, Julie A. Schneider, Rakez Kayed, Fabio Benfenati, Michael K. Lee, David A. Bennett, andView ORCID ProfileSylvain E. LesnéaDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;dRush Alzheimer s Disease Center, Rush University Medical Center, Chicago, IL 60612;eDepartment of Neurology, University of Texas Medical Branch, Galveston, TX 77555;fCenter for Synaptic Neuroscience, Istituto Italiano di Tecnologia, 16132 Genoa, Italy;gDepartment of Experimental Medicine, University of Genova, 16132 Genoa, ItalySee allHide authors and affiliationsPNAS June 6, 2017 114 (23) E4648-E4657; first published May 22, 2017; https://doi.org/10.1073/pnas.1704698114 Megan E. Larson aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteSusan J. Greimel aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteFatou Amar aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteMichael LaCroix aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteGabriel Boyle aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteMathew A. Sherman aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteHallie Schley aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteCamille Miel aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteJulie A. Schneider dRush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL 60612;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteRakez Kayed eDepartment of Neurology, University of Texas Medical Branch, Galveston, TX 77555;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteFabio Benfenati fCenter for Synaptic Neuroscience, Istituto Italiano di Tecnologia, 16132 Genoa, Italy;gDepartment of Experimental Medicine, University of Genova, 16132 Genoa, ItalyFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteMichael K. Lee aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteDavid A. Bennett dRush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL 60612;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteSylvain E. Lesné aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55414;bN. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, MN 55414;cInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN 55414;Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteORCID record for Sylvain E. Lesné For correspondence: lesne002@umn.edu Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 24, 2017 (received for review April 4, 2017) SignificanceAlzheimer’s disease (AD) is the most common form of dementia affecting an estimated 5.3 million Americans based on the 2015 Report of the Alzheimer Association. Our current understanding of the pathogenesis of AD suggests that soluble, nonfibrillar forms of amyloid proteins [e.g. amyloid-β, tau, and α-synuclein (αSyn)] may be responsible for impairing cognition and have therefore been advanced to be the most bioactive species in this brain disorder. We sought to determine the potential contribution of αSyn oligomers to AD-associated cognitive decline. We found that selective αSyn oligomers are elevated in AD brains and that genetically elevating oligomeric αSyn in an AD mouse model led to a selective decrease in presynaptic proteins and cognitive performance.AbstractMounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.α-synucleinoligomermemoryAlzheimer’s diseasesynapsinsAlthough abnormal protein aggregates in the form of amyloid plaques, neurofibrillary tangles, and Lewy bodies (LB) characterize neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease, an accumulating body of evidence indicates that soluble multimeric species of these proteins, also known as oligomers, might underlie the deleterious cascades of molecular changes ultimately resulting in these chronic brain disorders (1⇓–3). In this context, we define soluble endogenous amyloid oligomers as multimeric assemblies that (i) remain soluble in aqueous buffers following ultracentrifugation, (ii) are SDS-resistant following tissue lysis, (iii) are separated in liquid-phase chromatography, and (iv) are immunoreactive to at least two different antibodies for that amyloid molecule. Despite a well-accepted consensus that α-synuclein (αSyn) aggregation is critical for synaptic deficits, the exact relationship between various αSyn aggregation states and synaptic/cellular toxicity has not been formally established and remains a highly debated point of contention.The normal function of αSyn remains poorly understood (4). It appears to be regulating the size of the presynaptic vesicle pool (5) and assisting in the formation of the SNARE complex (6, 7). Supporting this concept, overexpression of human WT αSyn (h-αSynWT) was shown to inhibit vesicle release, presumably through a reduction in the synaptic vesicle recycling pool, and a selective lowering of complexins and synapsins (8). Moreover, large multimeric assemblies of recombinant αSyn were recently shown to inhibit exocytosis by preferentially binding to synaptobrevin, thereby preventing normal SNARE-mediated vesicle docking (9). This point might be particularly important because αSyn was suggested to interact with synapsins, because of their colocalization and common association with the recycling pool, and as synapsin-I mediates the binding of recycling vesicles to the actin cytoskeleton (10, 11).Beyond its physiological function, the native structure of αSyn has also been the subject of much debate (12⇓⇓–15). Briefly, the native state of αSyn was believed for a long time to be an unfolded ∼14-kDa monomer that only acquired an α-helical structure upon binding to lipids (16). However, several recent studies challenged this notion, claiming that the native state was primarily a folded tetramer of ∼58 kDa (15, 17). Given that the native tetramer was not prone to aggregation, these authors concluded that the oligomeric and fibrillar forms likely result from destabilization of the apparent αSyn tetramer (15). Other studies have, however, challenged the existence of a native tetrameric αSyn, concluding that endogenous αSyn purified from brain tissue consists of a largely unstructured monomer and is prone to aggregation (12, 14). Altogether, the consensus appears to be that αSyn may exist as monomers and soluble physiological multimers in cells, and that some forms of αSyn oligomers (o-αSyn) may be responsible for toxicity (4).We previously reported that the abundance of soluble, intracellular (IC) αSyn monomers was increased in AD brain tissue compared with controls in the absence of apparent LBs. Moreover, the amounts of IC monomeric αSyn in temporal cortices translated into a better biological correlate of AD-associated cognitive impairment than soluble amyloid-β (Aβ) and tau (18). Finally, the threefold overexpression of h-αSynWT was sufficient to trigger memory deficits in 7-mo-old transgenic (Tg)I2.2 mice in the absence of αSyn cytopathology (18). At a cellular level, the elevation of IC monomeric αSyn observed in AD coincided with selective reductions in two presynaptic proteins, synapsin and complexin, and with a perturbed colocalization of αSyn and synapsins within presynaptic vesicles (18), in agreement with previous reports (8, 19, 20). These observations therefore suggested a possible connection between the dysregulation of αSyn expression and alterations in presynaptic vesicle composition and release.It is in this uncertain context that we sought to determine whether an aberrant formation of o-αSyn accompanying the changes in monomeric αSyn previously seen in our AD cohort (18) was contributing to the decrease in synapsins and enhanced memory deficits. In the present study, we found that selective o-αSyn species accumulated in AD brain tissue in the absence of IC LB pathology. In particular, we observed that ∼28- to 35- and ∼56-kDa αSyn species (putative dimers and tetramers, respectively) were elevated by ∼1.5- to 2-fold, consistent with the 1.7- to 2.3-fold elevation previously documented for monomeric αSyn (18). Using biochemical and immunological approaches, we confirmed the oligomeric nature of these αSyn assemblies and observed that the abundance of o-αSyn species was inversely correlated with synapsins and cognitive function in our human cohort. To test whether elevating endogenous soluble h-αSynWT oligomers was sufficient to induce enhanced cognitive deficits and a selective reduction in synapsins in a mouse model of AD (21), we created a bigenic mouse line overexpressing mutant human amyloid precursor protein (APP) and h-αSynWT, with the intent that Aβ would promote the aggregation of αSyn (22). In this new APP/αSyn line, the oligomerization of αSyn was increased by fourfold, accompanied by a selective decrease of synapsins and by an exacerbation of Aβ-induced cognitive deficits. Finally, IC delivery of isolated endogenous o-αSyn, but not monomeric αSyn, in primary cortical neurons specifically triggered a decrease in synapsin expression. Overall, our data indicate that oligomeric αSyn-mediated lowering in synapsins might enhance AD-associated cognitive deficits.ResultsElevation of Soluble αSyn Oligomers in AD Brain.To determine whether o-αSyn might be elevated in parallel to the increase in IC monomeric αSyn previously reported (18), in the absence of LB pathology (Fig. S1), we created an in-house ELISA to detect soluble multimers of αSyn by homotypic recognition and soluble αSyn conformers immunoreactive to the A11 antibody (Fig. 1A). Using both detection sets, oligomeric αSyn species were elevated in AD subjects compared with age-matched, noncognitively impaired controls (NCI) by 34% and 48%, respectively (Fig. 1 B–D). Similar changes were detected in the mild cognitive impairment (MCI) group, albeit overall lower (Fig. 1 C and D). The selectivity and sensitivity of the assay was confirmed by comparing increasing concentrations of recombinant monomeric or oligomeric αSyn (rec-hαSynWT) and using brain lysates from WT, TgI2.2, and SNCA-null mice (Fig. S1). For a large proportion, the abundance of soluble αSyn assemblies was detected similarly well by the homotypic LB509-LB509 and heterotypic LB509-A11 sets, as indicated by the correlation observed between brain levels of LB509+ o-αSyn and A11+ o-αSyn species (Spearman’s ρ = 0.487, P 0.0001, n = 84) (Fig. 1E).Download figureOpen in new tabDownload powerpointFig. 1. Identification of soluble αSyn assemblies in human brain tissues. (A) Experimental design of oligomeric αSyn ELISA. The capture antibody consisted in the human specific αSyn antibody LB509 and a tandem of detecting antibodies (LB509-IR800 and A11-Biotin) was used to reveal oligomeric αSyn. (B) Representative infrared images of oligomeric αSyn measurements in Religious Orders Study specimens (n = 84) using either LB509-LB509 (homotypic) or LB509-A11 sandwiches on 96-well ELISA plates. Each well represents a separate patient sample. The dashed rectangle indicates increasing amounts of freshly resuspended recombinant monomeric αSyn (last row, samples 86–96, 1 pg to 10 ng). Please note that no signal was detected confirming the specificity of the assay. (C and D) Box plots for oligomeric αSyn species using either the homotypic LB509 sandwich (C) or the LB509-A11 sandwich (D). Obvious differences were observed between the AD group (n = 24) and the NCI (N) group (n = 26). (Mann–Whitney U test, F1, 47 = 4.9728 and F1, 47 = 5.0115, respectively; Student t test, ★P 0.05 vs. NCI.) (E) Regression analyses indicated a positive correlation between oligomeric αSyn species detected with either LB509/LB509 (x axis) or with LB509/A11 (y axis) (n = 84; Spearman rank correlation, P 0.0001). (F) Western blot (WB) analyses of soluble αSyn species in IC-enriched fractions using 4D6. Tg mice from the TgI2.2 line and recombinant human αSynWT were used as positive controls. (G and H) Box plots for monomeric (G) and putative oligomeric (H) αSyn species in the temporal cortex of subjects with NCI, MCI, or AD devoid of αSyn inclusions. Numbers in parentheses indicate group sizes. NCI is shown in green, MCI in blue, and AD in magenta boxes. In box plots of all figures, the bar inside the box indicates the median; the upper and lower limits of boxes represent the 75th and 25th percentiles, respectively. Bars flanking the box represent the 95th and fifth percentiles. (Kruskal–Wallis followed by Mann–Whitney U test, F1, 66 = 4.2289, F1, 66 = 4.4464, F1, 66 = 4.7717, F1, 66 = 3.8853, and F1, 66 = 2.4774 respectively; ★P 0.05 vs. NCI.) A.U., arbitrary units.Download figureOpen in new tabDownload powerpointFig. S1. Characterization of the human brain tissues and the o-αSyn ELISA used in this study. All tissue specimens used were previously characterized by histological analysis at Rush University. To confirm the initial characterization, 15 Religious Orders Study participants were randomly selected for confocal analysis and 5 are shown here. Numbers correspond to the Religious Orders Study identity number (ROS ID). (A and B) Representative confocal images of temporal neurons immunostained for total αSyn (4D6, magenta), pS129-αSyn (pS129, green), and the microtubule-associated protein-2 (MAP2; blue channel) confirmed the absence of LB inclusions in the ITG used in our study. Brain tissues previously characterized as displaying cortical LBs were used as positive controls. (n = 3 sections per case; n = 15). Arrowheads indicate neurons with αSyn aggregates. (Magnification: 20×; Insets, 60×.) (C) Comparative detection of αSyn monomers and oligomers using the homotypic o-αSyn ELISA. Each point corresponds to the average of three independent measurements using the 680- and 800-nm channels on a Li-Cor Odyssey platform. There was no apparent cross-reactivity with freshly resuspended αSyn monomers (up to 50 ng/mL). (D) Representative infrared images of oligomeric αSyn measurements in 11-mo-old WT, TgI2.2, and SNCA-null forebrain lysates using either LB509-LB509 (homotypic; 680 nm) or LB509-A11 (800 nm) sandwiches on 96-well ELISA plates. Each horizontal lane corresponds to increasing protein loads (0.1, 1, and 10 μg). A.U., arbitrary units.; Veh., vehicle.To determine the size of the putative soluble o-αSyn detected, we combined size-exclusion chromatography (SEC) with antibody detection under denaturing or native conditions. Western blotting analyses of isolated SEC fractions using IC-enriched lysates from AD individuals with normal (AD-normal) or high (AD-high) levels of αSyn (18) revealed the presence of various soluble αSyn species (Fig. S2). In contrast, freshly resuspended monomeric rec-hαSynWT did not behave as a globular protein in the SEC column because of its disordered structure, resulting in its elution at fractions 49–53 instead of fractions 61–63 (Fig. S3A), in line with earlier observations (12, 14, 15). AD brain-derived αSyn monomers eluted in fractions 43–53, likely because of the presence of two distinct 17- and 14-kDa monomeric forms (Fig. S2A). This finding was also observed in TgI2.2 mice overexpressing h-αSynWT (Fig. S3B). In addition, we noticed the presence of putative SDS-resistant αSyn species in Tg mouse and human brain lysates that appeared to behave as globular proteins (Figs. S2A and S3B). Several 4D6-immunoreactive bands of ∼28, 35, 56, and 72 kDa were readily detected, consistent with potential dimers and tetramers of the 14- and 17-kDa αSyn monomers mentioned above. Of note, the putative 28-kDa dimer eluted at its predicted globular molecular weight and did not coelute with any other detectable αSyn species (considering a detection limit for αSyn of ∼2.5 pg). This observation also argued against the possibility that this assembly was the result of a breakdown of a larger structure or of a self-aggregation of αSyn monomers. Quantitative densitometry analysis revealed significantly higher amounts of the 17- and 28-kDa species (a 1.42- and 2.23-fold elevation, respectively) in AD-high subjects compared with AD-normal (Fig. S2B), suggesting a differential elevation of o-αSyn in AD and intrinsically validating the results reported earlier by our group (18).Download figureOpen in new tabDownload powerpointFig. S2. Identification of soluble αSyn assemblies present in human brain tissues. (A) Western blot (WB) profile of soluble αSyn molecules detected in SEC fractions from brain tissue of subjects with normal (Upper) or high (Lower) levels of soluble αSyn. Blue arrows indicate the elution of used globular standards. Samples of interest were separated onto two separate 12-well gels in parallel. (B) Averaged signal intensity for the indicated apparent αSyn assembly detected in each SEC fraction obtained from both subgroups (the solid line represents the mean and shaded areas correspond to SDs). Abnormal elevations were detected for putative αSyn dimers (28 kDa) and monomers (17 kDa) in the brain tissues of AD-high subjects (Kruskal–Wallis followed by Mann–Whitney U test, ★P 0.05, n = 3 per SEC fraction; n = 5 per group). A.U., arbitrary units.Download figureOpen in new tabDownload powerpointFig. S3. SEC profiles of recombinant human αSyn monomers and of soluble αSyn assemblies present in TgI2.2, WT, and SCNA-null forebrain tissues. (A) Representative Western blot (WB) profile of recombinant human αSyn monomers detected in SEC fractions. Blue arrows indicate the elution of used globular standards. The Left Inset corresponds the nonsegregated material. Samples of interest were separated onto two separate 12-well gels in parallel. (B–D) Representative Western blot profile of soluble αSyn molecules detected in SEC fractions from forebrain tissue of 11-mo-old TgI2.2 (B), WT (C), and SNCA-null (D) mice. Of total proteins from the IC-enriched fraction, 250 μg was injected. The Left Inset in B corresponds with the nonsegregated material. Samples of interest were separated onto two separate 12-well gels in parallel.To further characterize the oligomeric nature of these soluble αSyn species, we performed nondenaturing analyses of SEC fractions by dot-blotting assay using IC-enriched extracts from AD-high, TgI2.2, WT, and SNCA-null mice (Fig. S4). Each fraction was subjected to a panel of commercially available antibodies detecting human αSyn (LB509, 4B12), mouse/human αSyn (4D6), phosphorylated and misfolded αSyn (pS129-αSyn and Syn514), and to a panel of antibodies generated to detect oligomeric and aggregated amyloid proteins (A11, OC, Officer), including o-αSyn (Syn33, F8H7) (23). We also included analyses with the 6E10 antibody detecting Aβ1–16 to determine whether the putative o-αSyn might be coupled to Aβ as a hybrid oligomer (24). Although a clear signal was detected in fraction 38, likely because of soluble APP or Aβ protofibrils, the pattern obtained with 6E10 was distinct from those found with αSyn antibodies. LB509 and 4B12 antibodies readily detected isolated monomeric αSyn in AD and TgI2.2 fractions, but not in either WT or SNCA-null fractions (Fig. S4A, lane 2). However, under these experimental settings, both proved quite poor at detecting αSyn in SEC fractions containing apparent o-αSyn (Fig. S4A, lanes 3–4). Using 4D6 modestly improved detection (Fig. S4A, lanes 3–4). We hypothesized that the 4D6 epitope was partly available because of the conformation of the putative o-αSyn species. To relax the folding of the protein, we boiled nitrocellulose membranes onto which samples had been previously preadsorbed. Under these new conditions, the detection of αSyn with 4D6 was substantially improved, revealing the presence of αSyn assemblies, consistent with 28-kDa dimers and cosegregated 35-kDa/72-kDa multimers (Fig. S4A). To confirm that these species corresponded to αSyn oligomers, we used antibodies detecting various oligomers of amyloid proteins (i.e., A11, OC, Officer) (25, 26), as well as antibodies specific to o-αSyn, Syn33, and F8H7 (23). OC and Officer detected fibrillar amyloid species cosegregating with the 35-kDa/72-kDa αSyn molecules in both AD and TgI2.2 samples, suggesting that the αSyn forms detected in SEC fraction 38 were prefibrillar oligomeric αSyn assemblies. In contrast, the 28-kDa αSyn dimers were detected with A11 and F8H7 in AD brain tissue and to a lesser extent in TgI2.2 mice, indicating that this αSyn species is indeed a nonfibrillar oligomer. Of note, we observed a faint immunoreactivity of SEC fraction 38 in the WT samples by antibodies 4D6 (boiled) and F8H7 (Fig. S4A), likely indicating the existence of physiological multimeric αSyn assemblies. As expected, the same analysis performed with SNCA-null mouse tissue did not yield any signal. In addition, SEC fractions containing the apparent 72-, 17-, and 28-kDa αSyn species isolated from either AD-normal or AD-high groups were subjected to the o-αSyn ELISA and confirmed the selective increase in discrete o-αSyn species (i.e., 28-kDa αSyn) (Fig. S4B).Download figureOpen in new tabDownload powerpointFig. S4. Nondenaturing analyses of soluble αSyn species isolated by liquid-phase chromatography. SEC fractions containing segregated soluble αSyn species isolated from brain tissues of AD subjects with high levels of αSyn (AD-high), or from mouse brain tissues (TgI2.2, WT, and SNCA-null mice) were analyzed under native conditions. (A) Dot blot analysis of SEC-isolated soluble αSyn molecules in AD-high specimen, 11-mo-old TgI2.2, WT, and SNCA-null mice (n = 3–6 per group per antibody) using commercially available antibodies against human αSyn (LB509 and 4B12), mouse/human αSyn (4D6), oligomeric αSyn (Syn33, F8H7), and aggregated amyloid proteins (A11, OC, and Officer). Finally, 6E10, a monoclonal antibody raised against human Aβ1–16 was used as an internal control. (B) ELISA analysis of SEC-isolated soluble αSyn molecules in AD specimens, 11-mo-old TgI2.2 mice and recombinant human αSyn monomers (n = 3–6 per group). (C) Relative fluorescence intensity corresponding to αSyn levels in SEC fraction tested. (Histograms represent the mean ± SD; Student t test, ★P 0.05, n = 6 per group.)To demonstrate that these 4D6-immunoreactive molecules corresponded to o-αSyn, we turned to TgI2.2 mice, a simpler model in which h-αSynWT is overexpressed. As previously reported, these animals did not display LB pathology (Fig. S5A). An age-dependent increase of the same αSyn species detected in AD brain tissue was observed in brain lysates of TgI2.2 mice at 4, 7, and 11 mo of age compared with WT and knockout littermates (Fig. S5 B and C). Of note, monomeric αSyn also increased with aging (Fig. S5D). We then subjected TgI2.2 IC fractions to hexafluoroisopropanol (HFIP) to promote the disassembly of putative o-αSyn into soluble αSyn monomers (Fig. S5 E and F). Low concentrations of HFIP (10–20%) appeared to trigger the oligomerization of low-molecular weight (LMW) αSyn species, as evidenced by the detection of larger species immunoreactive to 4D6 creating the appearance of a smear in the upper parts of the SDS/PAGE gel and by the reduction in the abundance of putative low-n o-αSyn. Increasing HFIP concentration to 100% induced the destruction of the quaternary structure of o-αSyn multimers into monomeric αSyn molecules (Fig. S5 E and F). It is worth noting that the 72-kDa band remained partially unaffected by this treatment, consistent with the detection of a faint band in IC protein lysates from SNCA-null mice (Figs. S3 and S5). Overall, these results indicate that the αSyn assemblies detected by 4D6 are indeed oligomeric in nature.Download figureOpen in new tabDownload powerpointFig. S5. Histochemical and biochemical characterization αSyn species detected in TgI2.2 mice. (A) Representative confocal images for MAP2 (blue) and αSyn (green) illustrating the cellular localization of αSyn in the hippocampi (CA1) of 7-mo-old WT and TgI2.2 littermates. Note the absence of apparent αSyn inclusions in transgenic animals. (Scale bars: 20 μm.) (B) Western blot (WB) analyses of IC fractions of 4-, 7-, and 11-mo-old WT, TgI2.2, and SNCA-null mice (KO) with 4D6 revealed the detection of putative αSyn assemblies of 28, 35, 56, and 72 kDa. Please note the absence of signal of αSyn in KO animals; only a faint nonspecific band was detected at ∼70 kDa. (C and D) Densitometry analyses revealed selective elevations in apparent αSyn oligomers (C) and monomers (D). (Histograms represent the mean ± SD; Student t test with Bonferroni correction, ★P 0.05 vs. 4-mo-old mice, ☆P 0.05 vs. 7-mo-old mice, n = 6 animals per genotype.) (E) Solvent-induced disassembly of putative brain αSyn oligomers. Representative Western blot analysis of TgI2.2 brain lysates subjected to increasing amounts of HFIP and revealed with 4D6. (F) Apparent soluble αSyn oligomers disassembled in 20% HFIP, with concomitant enrichment of monomeric αSyn (lower exposure provided in the Lower Inset of E for enhanced contrast). The data for fold-change in αSyn species corresponds to the mean ± SD. (ANOVA followed by Student t test with Bonferroni correction, ★P 0.05 vs. 0% HFIP, ☆P 0.05 vs. 20% HFIP, n = 3–4 per condition.) M, months.With the identification of 4D6 as the most sensitive antibody to detect o-αSyn under denaturing conditions (Figs. S4 and S5), we then reanalyzed the extracellular (EC)- and IC-enriched fractions of human brain specimens previously characterized using LB509 (18). As hypothesized, apparent SDS-resistant o-αSyn were readily detected by 4D6 in IC and EC fractions (Fig. 1 F and G and Fig. S6, respectively). In agreement with earlier results from our own group, we did not find differences in soluble EC αSyn monomers between clinical groups (Fig. S6 A and B). In contrast, soluble αSyn species of ∼17, 28, and 56 kDa were, respectively, elevated by 1.64-, 1.75-, and 1.64-fold in the IC fraction of AD subjects compared with NCI individuals (Fig. 1 G and H) and reduced in the EC fractions of these brain tissues (Fig. S6 A and B). Interestingly, a rise of the ∼56-kDa αSyn species was also detected in brain tissue from individuals diagnosed with MCI compared with NCI. Finally, these changes did not appear to be limited to the inferior temporal gyrus as other brain regions (angular gyrus, entorhinal cortex) also displayed elevations in apparent o-αSyn (Fig. S6 C–G). Clearly, larger studies will be needed in the future to extensively compare regional differences within each subject.Download figureOpen in new tabDownload powerpointFig. S6. Relative expression of soluble αSyn species detected in EC-enriched ITG lysates and in IC-enriched lysates from additional brain regions. (A) Western blot (WB) analyses of soluble αSyn species in EC-enriched fractions using 4D6. Transgenic mice from the TgI2.2 line were used as positive controls. (B) Quantification of monomeric and oligomeric αSyn species in the inferior temporal cortex of subjects with NCI (N), MCI (M), or AD. Numbers in parentheses indicate group sizes. NCI is shown in green, MCI in blue, and AD in magenta boxes. (C) Western blot analyses of soluble αSyn species in IC-enriched fractions from MF, AG, CALC, and enthorinal (EC) cortices using 4D6. Transgenic TgI2.2 and WT mice were used as positive and negative controls, respectively. (D–G) Comparative analysis of monomeric and oligomeric αSyn species detected in ITG, AG, CALC, enthorinal, and MF cortices of subjects with NCI, MCI, or AD. Italicized numbers in parentheses indicate group sizes. NCI is shown in green, MCI in blue, and AD in magenta boxes. In box plots of all figures, the bar inside the box indicates the median; the upper and lower limits of boxes represent the 75th and 25th percentiles, respectively. Bars flanking the box represent the 95th and fifth percentiles. (Kruskal–Wallis followed by Mann–Whitney U test, ★P 0.05 vs. NCI.) A.U., arbitrary units.Altogether, our data suggest that specific LMW o-αSyn species can accumulate in AD in absence of LB pathology.O-αSyn Species Negatively Correlate with Measures of Cognitive Performance.To determine whether the elevation in soluble o-αSyn species identified in AD brain tissue might be associated with cognitive deficits, we performed multivariable regression analyses using all measurements of soluble forms of αSyn detected with either 4D6 or LB509 and measures of cognitive function (Fig. 2). Analyzed cognitive domains included episodic, semantic, and working memory, visuospatial ability, perceptual speed, and global cognition. Following multivariate regression analyses, color maps for correlation indexes revealed that neither EC nor IC o-αSyn were correlated with cognition in aged-matched controls (Fig. 2A, Left color maps). In contrast, we observed that inferior temporal gyrus (ITG) levels of putative 28-, 35-, and 56-kDa o-αSyn were inversely correlated with episodic memory deficits (ρ = −0.661, P = 0.0376; ρ = −0.833, P = 0.0098, and ρ = −0.556, P = 0.0203, respectively) (Fig. 2A, Right color map). Overall, there was a generalized trend toward an inverse correlation between cognitive function and all IC αSyn species in our AD group. In agreement with our earlier studies (18), we found that levels of IC monomeric αSyn were inversely correlated to episodic memory and visuospatial ability, despite using a different antibody to measure αSyn (i.e., 4D6 instead of LB509).Download figureOpen in new tabDownload powerpointFig. 2. Soluble αSyn assemblies are associated with changes in cognitive function and synaptic expression in AD. (A) Following the measurements of soluble αSyn species in EC- and IC-enriched fractions of human temporal cortices, multivariate analysis was performed within the NCI and AD groups. Monomeric αSyn expression was used as positive control (18) and βSyn expression was used as negative control. Finally, all measures of proteins were performed using the same technique (SDS/PAGE followed by Western blot) to avoid inherent differences between techniques. Raw measurements of all proteins were used for the analysis. (Spearman’s ρ correlation with Bonferroni correction, ★P 0.05; ★★P 0.01, nNCI = 26 and nAD = 24). (B and C) Regression analyses between total synapsin protein expression and o-αSyn measured by ELISA using either LB509 (B) or A11 (C) as the detecting antibody in all AD cases tested (n = 24). Best-fitting models indicated significant negative correlations for both o-αSyn measurements (Spearman’s ρ, ρ = −0.346, P = 0.0241 and ρ = −0.551, P = 0.0052 respectively, n = 24). (D and E) Regression analyses between SYP protein expression and o-αSyn measured by ELISA using either LB509 (D) or A11 (E) as the detecting antibody revealed no correlations between o-αSyn and SYP (Spearman’s ρ, ρ = −0.1745, P = 0.4248 and ρ = −0.1470, P = 0.4932 respectively, n = 24). (F and G) Regression analyses revealed positive correlations between synapsin levels, total (F) or isoform specific (G), and episodic memory performance in our AD cohort (Spearman’s ρ, ρ = 0.4132, P = 0.0447 and ρ = 0.581, P = 0.0053 respectively, n = 24). A.U., arbitrary units.Brain Levels of Soluble αSyn Oligomers Correlate with a Selective Lowering in Synapsins I/II.The overexpression of h-αSynWT is associated with a selective reduction in synapsins and complexins in mice (8). Given that large o-αSyn species were recently proposed to inhibit the docking of synaptic vesicles (9) and that synapsins regulate synaptic transmission and plasticity, we hypothesized that the increase in o-αSyn measured in our AD cohort could be related to the decrease in synapsins reported earlier (18). Measuring synapsin protein expression by Western blotting (8, 18), we found that the abundance of o-αSyn measured by ELISA using LB509 as the detection antibody inversely correlated with total levels of synapsin isoforms (Ia/b and IIa/b) in the ITG (R2 = −0.346, P = 0.0241) (Fig. 2B). Similarly, A11+ o-αSyn amounts correlated with the lowering in synapsin expression in AD brains (R2 = −0.578, P = 0.0007) (Fig. 2C), although to a greater extent than LB509+ αSyn quantitatively. To assess whether these relationships were specific to synapsins, we performed additional regression analyses using the protein abundance of other presynaptic markers, such as synaptophysin (SYP). Consistent with previous reports, SYP protein levels were reduced in AD compared with age-matched controls (Fig. S7 A and B) and correlated with global cognition (Fig. S7C). However, no correlations were found between o-αSyn and SYP using either ELISA detection pairs (Fig. 2 D and E). These data suggest that the elevation of o-αSyn in AD might alter synapsin expression or turnover.Download figureOpen in new tabDownload powerpointFig. S7. Relationships between SYP, synapsin-IIa expression, and antemortem cognitive performance. (A) Western blot (WB) analyses of human SYP in the inferior temporal gyrus using membrane-associated (MB) extracts. Actin was used as internal loading control. (B) Quantification of the relative protein levels for SYP revealed a decrease in SYP abundance in AD vs. NCI (N) (Kruskal–Wallis followed by Mann–Whitney U test, ★P 0.05 vs. NCI). (C) Regression analysis between SYP expression and the global cognition composite index (Spearman’s ρ, ρ = 0.3388, P = 0.0009, n = 85). (D and E) Regression analyses between SYNIIa expression in the ITG and performance in various memory modalities, including semantic memory, working memory, perceptual speed, visuospatial memory, and global cognition. (Spearman’s ρ, n = 24.) A.U., arbitrary units; M, MCI.Oligomeric αSyn-Associated Lowering of Synapsin-I/II Correlates with Memory Impairment.We then determined whether the observed reduction in synapsins might be associated with episodic memory deficits, because this memory modality is specifically affected in AD (Fig. 2 F and G). We found that greater deficits in episodic memory correlated with lower total synapsin (-I/II) levels (Spearman’s ρ = 0.4132; P = 0.0447). Comparisons between synapsin isoforms (Ia, Ib, IIa, and IIb) further validated this trend as shown for SYN-IIa and episodic memory (Spearman’s ρ = 0.581; P = 0.0053) or other memory modalities (Fig. S7 D and E).We previously reported that 7-mo-old TgI2.2 mice present with spatial reference memory deficits (18). As shown in Fig. 3 and Figs. S3 and S5, despite the faint detection of o-αSyn in the forebrain of 3- to 4-mo-old TgI2.2 animals, the protein abundance of synapsin isoforms was similar to that of WT mice (Fig. S8 A and B). Other presynaptic proteins, such as complexins, Rab3, and SYP were also indistinguishable between genotypes at that age. However, at ages when TgI2.2 mice are cognitively impaired in the Barnes circular maze (BCM) (18), we observed a 30–40% reduction of synapsin proteins at 7 mo, compared with WT mice, and an exacerbation of these changes at 11 mo of age (Fig. S8 C and D). Furthermore, transcriptional analysis of SYN1, SYN2, CPLX1, CPLX2, SYP, and SYT mRNAs revealed a selective down-regulation of synapsin transcripts with aging in TgI2.2 mice (Fig. S8E). These findings suggest an association between αSyn, synapsin expression, and memory function.Download figureOpen in new tabDownload powerpointFig. 3. Genetic elevation of oligomeric αSyn in the J20 mouse model of Alzheimer’s disease is associated with a selective reduction in synapsin expression and exacerbated cognitive deficits. Three-month-old non-Tg WT, J20, TgI2.2, and J20×TgI2.2 mice were analyzed in the BCM. Immediately following behavioral testing, mice were killed for gene and protein analyses. (A) Representative Western blot images for transgene-derived human αSyn and total αSyn (mouse and human) using forebrain IC lysates. Actin was used as internal control. (B) Quantification of αSyn species revealed a significant elevation of putative o-αSyn in J20×TgI2.2 mice at 3 mo (ANOVA followed by Student t test with Bonferroni correction, F3, 24 = 754.193, ★P 0.05 vs. WT, ☆P 0.05 vs. TgI2.2, n = 6 per age per genotype). (C) Representative Western blot images for synapsins and SYP using forebrain MB lysates. Actin was used as internal control. (D) Densitometry analyses confirmed the apparent visual reduction in synapsins in bigenic J20×TgI2.2 mice compared with other mouse groups (ANOVA followed by Student t test with Bonferroni correction, ★P 0.05 vs. WT, n = 6 per age per genotype). (E) Double labeling for αSyn (green) and synapsins (magenta) in 6-μm-thick sections of the CA1 domain of the hippocampus from 3-mo-old WT and J20×TgI2.2 mice. (Scale bars: 20 μm, Upper; 4 μm, Lower.) (F) Quantification of the colocalization between αSyn/SYN in the stratum radiatum of WT, J20, TgI2.2, and J20×TgI2.2 mice using Bitplane’s Imaris7.x colocalization tool. Z-stacks of images were transformed for volume rendering and voxel count analysis was performed. (Histogram values represent mean ± SD, ANOVA followed by Student t test with Bonferroni correction, F3, 48 = 167.576 and, F3, 48 = 64.229, ★P 0.05 vs. WT, n = 6 animals, 8 fields per mouse.) (G) Three-month-old non-Tg C57BL/6, J20, TgI2.2, and J20×TgI2.2 mice were trained in the BCM for 4 d. A probe trial (escape platform removed) was conducted 24 h after the last training session. During acquisition of the task, escape latency to complete the task was recorded. Although J20 and TgI2.2 groups learned this task comparably to WT mice, J20×TgI2.2 bigenic mice displayed a severe acquisition deficit. In these mice, two-way repeated-measures ANOVA (RMANOVA) revealed an effect of transgene (F = 36.89, P = 0.0008) but no significant effect of training (F = 8.02, P = 0.8236). Although different from WT animals, J20×TgI2.2 mice were partly able to learn the task (★P 0.05 vs. WT mice). (H) During the probe trial, J20×TgI2.2 animals did not elicit a spatial search bias compared with WT and single Tg littermates. Bigenic J20×TgI2.2 mice consistently performed worse than age-matched single Tg J20 and TgI2.2 animals (two-way ANOVA, ★P 0.05 vs. WT mice; ☆P 0.05 vs. J20×TgI2.2 mice). Data represent mean ± SEM (n = 6–8 males per age per genotype). (I) Relationship between probe trial performance and relative synapsin-IIa expression in all animals tested. The best fit is represented on the dot plot (R2 = 0.9111, P 0.0001, n = 24). (J) Regression analyses between probe trial performance and relative synapsin-IIa expression by genotype of tested animals revealed linear relationships within each group, including bigenic J20×TgI2.2 mice (R2 = 0.8659, P 0.01, n = 6 animals). A.U., arbitrary units.Download figureOpen in new tabDownload powerpointFig. S8. Age-dependent expression of synapsins in TgI2.2 mice. (A) Representative Western blot analyses of synapsins, complexins, Rab3 in MB fractions of 4-mo-old WT and TgI2.2 mice. Actin was used as internal loading control. (B) Quantification of the relative expression levels of synapsins, complexin isoforms, Rab3, and SYP in the forebrain of young WT and TgI2.2 animals. (ANOVA followed by Student t test with Bonferroni correction, n = 5–6 animals per group.) (C) Representative Western blot (WB) analyses of synapsin-I/II isoforms in MB fractions of TgI2.2 mice at 4, 7, and 11 mo of age. Actin was used as internal loading control. (D) Quantification of the relative expression levels of synapsin isoforms with aging in the forebrain of TgI2.2 animals (ANOVA followed by Student t test with Bonferroni correction, ★P 0.05 vs. WT, n = 4–6 animals per group). M, months.Enhancing αSyn Oligomerization in an AD Mouse Model Alters Synapsin Expression and Synaptic Localization.Because both monomeric and oligomeric αSyn increase with age in TgI2.2 mice (Fig. S4), thereby preventing the identification of putative changes linked to one or the other, we created a bigenic mouse line by crossing J20 mice (21) with TgI2.2 mice (27) to test whether an elevation in soluble h-αSynWT oligomers was sufficient to induce a selective reduction of synapsins in a mouse model of AD. Because Aβ and αSyn are known to promote the aggregation of each other in vivo (22, 28), we expected to trigger the oligomerization of αSyn when Aβ is overexpressed. We observed that the expression of transgene-derived h-αSynWT monomers was similar between TgI2.2 and J20×TgI2.2 mice (Fig. 3 A and B). However, we noticed a 3.7-fold increase in LMW o-αSyn in bigenic mice compared with TgI2.2 at 3 mo of age (Fig. 3 A and B). Importantly, we did not observe expression changes in APP and Aβ in these animals (Fig. S9 A and B) nor formation of amyloid deposits (Fig. S9C). This specific profile allowed us to test whether the ∼fourfold elevation in o-αSyn was associated with a selective decrease in synapsins. Although no overt changes in SYP and complexins were observed across all mouse genotypes, a reduction in synapsins was readily visible in J20×TgI2.2 bigenic mice compared with J20, TgI2.2, and WT animals (Fig. 3C). Densitometry analysis revealed significant decreases of synapsin Ia, IIa, IIIa, and IIb in the forebrain of bigenic mice with no apparent changes in SYP or complexins (Fig. 3D).Download figureOpen in new tabDownload powerpointFig. S9. Relative forebrain expression of APP, Aβ, αSyn, and synapsins in 3-mo-old mice used in the study. (A) Western blot (WB) analyses of human APP and total Aβ in forebrains of young WT, J20, TgI2.2, and J20×TgI2.2 mice using 6E10. Actin was used as internal loading control. (B) Quantification of the relative protein levels for APP and Aβ. (Histograms represent the mean ± SD; Student t test with Bonferroni correction, ★P 0.05 vs. J20, n = 6 group per genotype.) (C) Representative confocal images of CA1 hippocampal neurons immunostained for the MAP2 (blue channel), αSyn (4D6; green), and synapsins (magenta) revealed no aberrant formation of LB inclusions in 3-mo-old TgI2.2 and J20×TgI2.2 mice. A subtle global reduction in the signal for SYN could also be noticed (n = 6 sections per animals; n = 3–6 animals per genotype). (Scale bars: 20 μm.) M, months.We and others have previously reported that when αSyn is accumulating, its colocalization with synapsins at synaptic boutons is altered (8, 18, 20). However, the potential contribution of o-αSyn to this phenomenon is unknown. We therefore examined the colocalization of synapsins with αSyn in the stratum radiatum of the CA1 region of the hippocampus in 3-mo-old WT, J20, TgI2.2, and J20×TgI2.2 mice (Fig. 3 E and F). Confocal image analysis revealed a ∼40% reduction in the colocalization of αSyn with synapsins in bigenic animals compared with controls (Fig. 3F).Overall, these findings suggest that o-αSyn might selectively regulate the expression, cellular targeting, and turnover of synapsin proteins.Elevating o-αSyn Exacerbates Memory Deficits in APP Mice.To determine whether the increase in o-αSyn and its associated decrease in synapsins exacerbated Aβ-induced cognitive deficits, we subjected all four animal groups to behavioral testing using the BCM to assess spatial reference memory at 3 mo of age (Fig. 3G). Bigenic J20×TgI2.2 animals displayed an apparent delay in learning the task compared with WT and single Tg J20 and TgI2.2 littermates (Fig. 3G). During the retention trial on day 5, J20×TgI2.2 mice did not show a search bias to the target hole whereas all other age-matched groups performed similarly (Fig. 3H). Regression analyses revealed positive correlations between synapsin isoform expression and memory integrity, as exemplified by results obtained for synapsin IIa across all animals (Fig. 3I) or within genotype (Fig. 3J). These results suggest that learning and spatial memory recall were affected in plaque-free J20×TgI2.2 mice in presence of elevated o-αSyn.IC Delivery of Exogenous o-αSyn Lowers Synapsin Protein Abundance.To demonstrate that o-αSyn were responsible for altering synapsin protein abundance, we sought for means to deliver o-αSyn isolated from brain tissues inside primary cortical neurons. Using the shuttling reagent Chariot (Active Motif), we first successfully established the principle that we could deliver large molecules: for example, fluorescently labeled antibodies, intracellularly (Fig. S10A). We then prepared preparations of rec-hαSynWT, which were segregated by SEC to obtain preparations enriched in αSyn monomers or oligomers (Fig. S10B). Six hours postdelivery, primary neurons that received rec-hαSynWT monomers displayed enhanced expression of monomeric αSyn, whereas cells that received rec-hαSynWT oligomers readily contained o-αSyn without noticeable changes in cell-derived αSyn monomers (Fig. S10C). Under these experimental conditions, no apparent changes in synapsin abundance were observed upon IC delivery of rec-hαSynWT (Fig. S10 D and E). Because the folding of αSyn might differ in vitro compared with that occurring in vivo, we isolated soluble αSyn species from 11-mo-old TgI2.2 mice by SEC (Fig. 4A). We selected to test whether fractions enriched in αSyn monomers (#48), low-n oligomers (#56), or larger oligomers (#38) could lower synapsin protein abundance in vitro. To confirm the absence of multimeric assembly in SEC fraction #48, we performed independent nondenaturing analyses of SEC fraction #48 by Clear Native-PAGE, in which we did not observe the presence of multimeric species (Fig. S10F). Vehicle, fractions that do not contain αSyn (#36) or corresponding fractions from SNCA-null mice were used as negative control. Immunofluorescence labeling of exogenous αSyn species confirmed the IC delivery of h-αSyn into cultured primary neurons (Fig. 4B). Accordingly, we found that the protein amounts of synapsin-I and -II were not changed in cells that received intraneuronal delivery of αSyn monomers (#48) compared with vehicle-treated neurons (Fig. 4 C and D). However, both fractions enriched in o-αSyn (#38 and #56) induced a ∼40% lowering in synapsin abundance 6 h postdelivery, reminiscent of the ∼40% reduction seen in the forebrains of 3-mo-old J20×TgI2.2 animals. In contrast, matching SEC fractions derived from SNCA-null mice did not lead to significant changes in synapsin protein amounts (Fig. 4 C and D).Download figureOpen in new tabDownload powerpointFig. 4. Intraneuronal delivery of oligomeric αSyn lowers synapsin expression in primary cortical neurons. (A) Representative Western blot image illustrating the detection of αSyn species following SEC separation of IC forebrain lysates of 9-mo-old TgI2.2 animals. Increasing amounts of recombinant human αSynWT was used as internal standard. (B) Immunofluorescent labeling of exogenous human αSyn (magenta) delivered intracellularly into cultured primary neurons 6 h postproteotransfection. Nuclei were labeled with DAPI (blue). (Scale bar: 10 μm.) (C) Representative Western blot analyses of synapsin isoforms in primary WT (Upper) and SNCA-null (Lower) cortical neurons exposed to SEC fractions containing oligomeric (#38 and #56) or monomeric (#48) αSyn derived from TgI2.2 mice for 6 h. Actin was used as internal standard. (D) Densitometry analyses revealed apparent reduction in synapsins in cells treated with oligomeric αSyn but not with monomeric αSyn. (Histogram values represent mean ± SD, ANOVA followed by Student t test with Bonferroni correction, F4, 50 = 24.930, F4, 50 = 0.819, and F4, 50 = 26.742 for WT cells + TgI2.2 fractions, WT cells + SNCA-null fractions and SNCA-null cells + TgI2.2 fractions respectively; ★P 0.05 vs. WT, n = 6 animals per group per genotype.) Veh., vehicle.Download figureOpen in new tabDownload powerpointFig. S10. Protein expression of synapsins, CREB, and Nurr1 in mouse primary cortical neurons following IC delivery of recombinant h-αSynWT species. (A) IC delivery of exogenous fluorophore-conjugated antibodies using Chariot in primary cortical neurons 60 min postapplication. Neurons were labeled with MAP2 (magenta). (Scale bars: 10 μm.) (B) Representative Western blot (WB) images for αSyn in selected fractions following SEC segregation of recombinant monomers and multimers. Fraction #50 was enriched in monomers, whereas #42 was enriched in αSyn multimers. (C) Representative Western blot images for αSyn using lysates of cells subjected to IC delivery of fractions #50 and #42 (250 nM, monomer equivalent). (D) Representative Western blot analysis of synapsin expression in primary neurons 6 h after intraneuronal delivery of recombinant monomers or oligomers isolated by SEC. Actin was used as internal loading control. (E) Quantification of the relative expression levels of synapsins following intraneuronal delivery of recombinant h-αSynWT species. (ANOVA followed by Student t test with Bonferroni correction, n = 4–6 dishes per group.) (F) Western blot analysis with 4D6 antibodies following Clear Native (CN)-PAGE segregation of SEC fraction #48 using IC lysates from TgI2.2, WT, or SNCA-null (KO) mice. Oligomeric recombinant h-αSynWT (0.25 μg) was used as control. (G) Representative Western blot images documenting the protein abundance for pS133-CREB, total CREB, Nurr1, and the neuronal nuclear protein NeuN in membrane lysates from primary neurons treated with αSyn species delivered with Chariot. Please note the selective reductions in phosphorylated (p)CREB and Nurr1, whereas NeuN amounts remain unchanged. Veh., vehicle.To test whether the exogenous αSyn assemblies transferred into cells required endogenous αSyn to lower synapsin protein amounts, we repeated these experiments using SNCA-null primary neurons. In this context, large αSyn species were not able to able to alter the normal synapsin protein profile, whereas the ∼28-kDa αSyn assembly still lowered synapsins (Fig. 4 C and D). These findings therefore suggest the presence of different functional conformers of αSyn with species requiring template assembly and another that did not. These results also directly demonstrated that o-αSyn selectively reduce synapsin proteins in neurons through an unknown mechanism.αSyn Oligomers Inhibit cAMP Response Element Binding- and Nurr1-Controled Transcription of SYN Genes.Finally, to assess whether o-αSyn alter SYN1 and SYN2 genes encoding for synapsin-I and -II, we measured the expression of transcripts for SYN1, SYN2, CPLX1, and CPLX2 (complexins), SYP, and SYT1 (synaptotagmin-I) by real-time quantitative PCR (rt-qPCR) in the forebrain of WT and TgI2.2 mice. Three ages were tested (i.e., 4, 7, and 11 mo of age), because we observed an age-dependent increase of o-αSyn during this period (Fig. S6). These analyses did not reveal transgene-driven differences in mRNA expression for any of the genes tested in the youngest group of animals. However, SYN1 and SYN2 mRNAs were selectively reduced by ∼20–30% at 7 and 11 mo of age in TgI2.2 mice compared with non-Tg littermates (Fig. 5A). To demonstrate that o-αSyn were directly responsible for this change, we introduced αSyn monomers (#48) or αSyn oligomers (#38 and #56) into cultured cortical neurons using Chariot-mediated delivery and measured the expression of SYN1, SYN2, CPLX1, CPLX2, and SYP mRNAs 6 h postdelivery. Reminiscent of the in vivo findings, SYN1 and SYN2 transcripts were down-regulated by 30–50%, whereas CPLX1, CPLX2, and SYP mRNAs were unchanged (Fig. 5B). Using the MRC DBD:Transcription factor prediction database (www.transcriptionfactor.org), we identified putative responsive elements for cAMP response element binding (CREB) and Nurr1 in the 5′UTR/promoter region of SYN1 and SYN2 (Fig. 5C), two transcription factors known to be suppressed by αSyn (29, 30). Importantly, these sequences are conserved between mouse and human genomes. We then measured the abundance of Nurr1 and the activated form of CREB phosphorylated at serine 133 (pS133-CREB) in forebrain tissues of TgI2.2 mice and found age- and transgene-dependent reductions in both transcription factors between 4 and 11 mo of age (Fig. 5 D–F). We also confirmed that IC delivery of o-αSyn in neurons led to a decrease in pS133-CREB and Nurr1 proteins (Fig. S10D). Finally, we performed gene promoter reporter assays in HEK293 cells to demonstrate that CREB and Nurr1 control the transcriptional expression of SYN1 and SYN2 genes, respectively. Using the dual luciferase system, we found that forskolin-induced CREB activation up-regulated both mouse and human SYN1 proximal promoter activities (Fig. 5G) and that expressing Nurr1 in cells elevated the activity of the human SYN2 proximal promoter (Fig. 5H). Taken together, these results suggest that o-αSyn selectively down-regulate SYN1 and SYN2 gene expression by inhibiting CREB and Nurr1.Download figureOpen in new tabDownload powerpointFig. 5. αSyn oligomers down-regulate SYN1 and SYN2 gene expression through CREB and Nurr1. (A) Age-dependent changes of SYN1, SYN2, CPLX1, CPLX2, SYP, and SYT1 gene expression by rt-qPCR analysis in the forebrain of TgI2.2 mice. Two-way ANOVA revealed a significant effect of transgene (F = 37.18, P 0.0001), of age (F = 21.09, P 0.0001), and transgene × age interaction (F = 4.92, P = 0.033) for SYN1 mRNA. The same analysis revealed a significant effect of transgene (F = 36.61, P 0.0001), of age (F = 18.37, P 0.0001), and transgene × age interaction (F = 4.37, P = 0.041) for SYN2 mRNA. (Histogram values represent mean ± SD, ANOVA followed by Student t test with Bonferroni correction, F3, 35 = 20.026 and F3, 35 = 18.99 for SYN1 and SYN2, respectively; ★P 0.05 vs. WT, n = 6–10 animals per age.) (B) Changes of SYN1, SYN2, CPLX1, CPLX2, SYP, and SYT1 gene expression by rt-qPCR analysis in primary cortical neurons following Chariot-mediated delivery of with isolated αSyn species. (Histogram values represent mean ± SD, ANOVA followed by Student t test with Bonferroni correction, F3, 24 = 9.173, P = 0.0004 and F3, 24 = 6.407, P = 0.0013 for SYN1 and SYN2, respectively; ★P 0.05 vs. WT, n = 6 dishes per treatment.) (C) Predicted response elements for CREB (light pink) and Nurr1 (dark pink) within mouse SYN1 and SYN2 genes. (D) Representative Western blot images illustrating the abundance of pS133-CREB, total CREB, Nurr1, and actin in forebrain lysates of 4-, 7-, and 11-mo-old WT and TgI2.2 mice. (E and F) Densitometry analyses revealed age-dependent reductions in the phosphorylated (p)CREB/CREB ratio (E) and in Nurr1 (F) protein amounts. Two-way ANOVA revealed a significant effect of transgene (F = 168.67, P 0.0001), of age (F = 129.39, P 0.0001), and transgene × age interaction (F = 72.74, P 0.0001) for the pCREB/CREB ratio. The same analysis revealed a significant effect of transgene (F = 91.34, P 0.0001), of age (F = 22.09, P 0.0001), and transgene × age interaction (F = 29.93, P 0.0001) for Nurr1. (Histogram values represent mean ± SD, two-way ANOVA followed by Student t test with Bonferroni correction, F3, 30 = 130.886 and F3, 30 = 49.870 for pCREB/CREB and Nurr1, respectively; ★P 0.05 vs. WT, ☆P 0.05 vs. 4-mo-old TgI2.2 mice, n = 5–6 animals per group per genotype.) (G and H) Dual luciferase gene promoter reporter assay revealed that the activity of mouse and human SYN1 (G) and SYN2 (H) promoters is positively modulated by CREB and Nurr1, respectively. Treating cells with 10 µM forskolin activated CREB as assessed by phosphorylation at S133 and nuclear translocation. (Histogram values represent mean ± SD, ANOVA followed by Student t test with Bonferroni correction, F5, 57 = 120.67 and F5, 56 = 112.48 for SYN1 and SYN2 promoters, respectively; ★P 0.05 vs. empty vector, ☆P 0.05 vs. stimulated cells, n = 10–12 dishes per group.) M, months; Veh., vehicle.DiscussionA common effort in the field of neurodegenerative diseases is to determine the pathogenic contribution of misfolded proteins once aggregation occurs. With this focus, there has been a paradigm shift toward studying the contribution of soluble, nonfibrillar forms of amyloid proteins, as these assemblies have been proposed to be more toxic than fibrillar species. In AD, intense focus has been set on early aggregates of Aβ and tau, as the fibrillary forms of these proteins have constituted the pathological hallmarks of the disease. Several years ago, however, we reported that disturbances in the abundance of another aggregation-prone amyloid protein, αSyn, might also be involved in AD pathophysiology (18). Because monomeric forms of amyloid proteins are prone to aggregation and because some soluble oligomeric assemblies of αSyn have been reported to be neurotoxic (31⇓⇓⇓–35), we hypothesized that the observed increase in apparent soluble αSyn monomers (18) was accompanied by an elevation of toxic αSyn oligomers, causing the observed decrease in synapsins and the exacerbation of memory deficits triggered by human Aβ and tau.Distinct Soluble αSyn Species Linked to AD-Associated Impairment.In this follow-up study, we identified putative multimers of two monomeric αSyn species of 14 and 17 kDa that included putative dimers (∼28 and 35 kDa), and tetramers (∼56 kDa) using a combination of biochemical techniques. The recognition of this pattern suggests the possible existence of two likely pathways for the aggregation of αSyn in vivo, a principle first suggested by molecular modeling of αSyn aggregates (36) and recently integrated into the proposed mechanisms of αSyn aggregation and propagation (3). This hypothesis is further supported by the existence of divergent detections of o-αSyn by the homotypic LB509 and heterotypic LB509-A11 pairs used for the ELISA studies, as recently reported for Aβ (37). If correct, the human samples showing detection with both homotypic and heterotypic pairs would contain both conformers. The disease significance to this observation is unclear at this time because of the small numbers of brain specimens composing all three categories (LB509+, A11+, and LB509/A11+) and because of the creation of necessary cut-offs, but certainly warrants larger studies to examine the functional role of these entities of αSyn. Although other groups have provided evidence that ∼35-kDa SDS-resistant αSyn dimers can be detected in brain tissue (24, 38, 39), we speculate that the unique experimental biological specimens used (i.e., human brain tissue with elevated expression of αSyn combined with an absence of LB pathology) allowed us to detect apparent multimers of 14- and 17-kDa αSyn monomers.We also believe that the detection of these various soluble forms was only possible using the antibody 4D6, which we recognized to display enhanced sensitivity toward o-αSyn following relaxation or denaturation of αSyn molecules, even compared with antibodies specifically raised to detect o-αSyn, such as Syn33 and F8H7. With these conditions, biochemical evidence suggested that o-αSyn accumulated intracellularly in AD brains compared with age-matched controls, whereas EC o-αSyn species were less abundant in the AD group. A possible interpretation of these results consists in o-αSyn being expelled from the cytosol of neurons under normal conditions, perhaps as a self-regulated protective mechanism, thereby creating an equilibrium between IC and EC compartments. In AD, this balance would be disrupted, facilitating the intraneuronal accumulation of αSyn oligomers. Given the emerging focus on prion-like spreading of amyloid aggregates, it remains to be determined whether the species studied here can propagate from cell-to-cell via the EC space as it was reported for dissociated fibrillar assemblies (40, 41).In addition to the accumulation of an ∼56-kDa αSyn species in IC fractions of AD brain tissues, the levels of this possible tetramer of the 14-kDa monomeric αSyn were inversely correlated to episodic and semantic memory performance. Although the exact structure and folding of endogenous αSyn remains highly controversial (12, 14, 15), we posit that the ∼56-kDa αSyn assembly detected in our studies is unlikely to correspond to the ∼55- to 60-kDa tetrameric αSyn first identified by the Selkoe group (13, 15, 17) because their biophysical properties appear different (notably their relative stability in presence of SDS) (42). If it were the case nonetheless, our findings suggest that an abnormal accumulation of these so-called \"physiological multimers” might be deleterious for neuronal function and cognition.Finally, on this topic, it is worth stressing that monomeric αSyn also correlated with cognitive function, and could therefore be a determining factor in modulating cognition as well.Synapsin-I/II Lowering and Cognitive Deficits in AD and Animal Models.Moreover, we documented that the amounts of o-αSyn detected under native conditions positively correlated with reductions in synapsin abundance in AD brain specimens. This relationship appeared to be relatively specific to the o-αSyn/synapsins pair, as similar analyses with SYN did not reveal an association with soluble o-αSyn levels.We also found that total synapsin-I/II levels were correlated to the level of episodic memory within the Religious Orders Study AD cohort examined. Our results are consistent with existing reports showing that reduction in synapsin-I gene expression induced by increased DNA methylation is linked to cognitive aging in rodents (43) and that ablation of either the synapsin-I or synapsin-II gene causes age-dependent cognitive impairment in mice involving emotional and spatial memory (44). Of note, 12- to 14-mo-old SYN1-null mice display neuronal loss and gliosis in the hippocampus and neocortex (44) further highlighting the importance of putative changes in synapsin expression in AD. In addition, SYN1 and SYN2 loss-of function mutations in humans were recently shown to be causative for autism spectrum disorder (45, 46), associated with excitatory/inhibitory imbalance and epileptic seizures (47). Strikingly, both of these changes have also emerged as prominent features of mouse modeling AD (48, 49) and early AD (50). Considering the results presented in this study, our previous results showing a ∼60% reduction in synapsins associated with the increase in soluble αSyn species in AD (18) and the growing recognition of network disturbance associated with AD (49⇓–51), we postulate that the lowering of synapsins-I and -II observed in AD might mediate the enhancement of memory deficits triggered by o-αSyn.To determine whether an elevation in soluble h-αSynWT oligomers could induce a selective reduction in synapsins in a mouse model of AD, we generated a bigenic mouse line expressing human Aβ and h-αSynWT, based on earlier observations indicating that Aβ and αSyn can potentiate the aggregation of each other in vivo (22). At 3 mo of age when pathological lesions are absent, forebrain o-αSyn were increased by ∼fourfold in J20×TgI2.2 bigenic mice compared with TgI2.2 littermates, but monomeric αSyn was unchanged. This marked rise of o-αSyn was associated with selective reductions in synapsin-I/II proteins and with profound deficits in learning and memory retention. This observation contrasts with earlier results showing that memory retention in 6-mo-old bigenic hAPP/hSYN (J9×D line) and in hAPP mice were identical (22). We speculate that this apparent discrepancy between these two studies might be because of the fact that hSYN-line D mice elicit LB inclusions as early as 3 mo of age and that motor function is compromised in bigenic hAPP/hSYN at 6 mo (52). Conversely, TgI2.2 mice do not develop pathological lesions (27) and do not display apparent motor deficits during behavioral testing as assessed by animal speed and distance run during the task.In this context, it is worth stressing a couple of important points related to the animal models used. First, recent studies reported that the overexpression of the A30P mutant of human αSyn in APP/PS1 Tg mice, another model of AD, led to a lowering of Aβ deposition and to synaptic abnormalities suggestive of synapse loss (53). The alterations in synaptic proteins observed in 3-mo-old bigenic J20×TgI2.2 mice appear reminiscent of those reported by Bachhuber et al. despite qualitative differences. However, our results indicate that overexpression of h-αSynWT worsens cognitive deficits in absence of deposited Aβ and αSyn, although it remains unknown whether the changes reported for APP/PS1 × αSynA30P bigenic animals translate into cognitive deficits. Furthermore, it also remains unclear whether h-αSynWT can alter Aβ-induced phenotypes, considering the distinct properties of αSynA30P (54). Second, despite previous evidence reporting the association of Aβ oligomers and cognitive deficits in J20 mice before plaque formation (55), the in vivo results presented here cannot rule out the possibility that the potentiation of the memory impairments seen in J20×TgI2.2 bigenic mice is a result of the overexpression of APP in these mice (i.e., ∼threefold over endogenous APP). However, recent analyses using single-cell qPCR revealed that individual neurons in sporadic AD can harbor an averaged copy number for APP of 3.8–4 (up to 12 copies) over control samples (56), suggesting that the threefold elevation of APP seen in J20 mice might actually be relevant to AD. That said, future studies using newly described APP knockin animals will be needed to address whether the overexpression of αSyn can also alter the phenotype of these lines (57).Overall, our genetic experiment replicated the changes observed in AD brain tissue and supports the notion that an increase in o-αSyn is associated with synapsin-I/II lowering and the potentiation of Aβ-induced cognitive impairment. It is also important to note that the J20×TgI2.2 bigenic model was created to selectively enhance αSyn oligomerization, and as such might not reproduce changes mediated by increased levels of αSyn transcripts and of the monomeric protein. Future studies with new animal models will be necessary to fully address their role in AD.Dependence on Endogenous αSyn for Exogenous o-αSyn to Induce Reductions in Synapsin Abundance.To directly demonstrate that o-αSyn induced a selective reduction in synapsin protein abundance, we used an in vitro approach to deliver exogenous αSyn species isolated from brain tissue of cognitively impaired TgI2.2 mice into primary neurons. Although intraneuronal delivery of exogenous αSyn monomers did not alter synapsin-I/II protein amounts compared with vehicle or to equivalent SEC fractions using SNCA-null lysates, IC delivery of o-αSyn fractions in cultured neurons caused a 40–50% reduction in synapsins-I and -II within 6 h. Because of accumulating evidence reporting prion-like propagation of small fibrillar amyloid aggregates (58⇓⇓–61), we asked whether endogenous αSyn was required for the tested exogenous o-αSyn to perturb synapsins-I and -II protein abundance. Our results indicated that larger o-αSyn species lost their ability to reduce synapsins when introduced into SNCA-null neurons lacking αSyn, whereas smaller o-αSyn assemblies did not depend on endogenous αSyn to lower synapsins, reminiscent of prion-like mechanisms. In agreement with earlier reports (35, 40, 62), these findings therefore suggested the presence of different functional conformers of αSyn with species requiring template assembly and others that did not. On the one hand, the fact that both αSyn aggregates trigger the same cellular change is surprising, as one would perhaps predict differential alterations in neuronal biology induced by each species. On the other hand, this apparent conversion on synapsin regulation could be viewed as a central and essential mechanism induced by soluble αSyn aggregates. As new tools and additional assemblies are isolated, future studies should be able to directly address this hypothesis.o-αSyn Down-Regulate SYN1 and SYN2 Gene Expression Through CREB and Nurr1.Finally, at a molecular level, we revealed that the reduction in synapsin proteins occurs in parallel to a selective down-regulation of the transcription of SYN1 and SYN2 genes in vivo when oligomeric αSyn species are present and in vitro when isolated αSyn oligomers are introduced intraneuronally. These results are consistent with the publicly available RNA sequencing data (NCBI Gene Expression Omnibus accession no. GSE70368) from recent studies using mouse primary midbrain neurons infected with αSyn (30). Upon αSyn overexpression, transcripts for SYN1, SYN2, and SYN3 were down-regulated by ∼30–70%, whereas CPLX1, CPLX2, SYP, and SYT1 mRNAs were unchanged compared with control neurons. Although the presence of o-αSyn was not disclaimed in these studies, we advance that these changes might be a result of the presence of αSyn oligomers based on our own in vitro results. Using predictive databases, we found that human and mouse SYN1 and SYN2 promoters contained conserved putative responsive elements for CREB and Nurr1, respectively, two transcription factors involved in αSyn-mediated toxicity (29, 30, 63). We then found that the protein abundance of pS133-CREB and Nurr1 was decreased in association with the age-dependent accumulation of αSyn oligomers in TgI2.2 mice and following intraneuronal delivery of o-αSyn. Finally, we confirmed that CREB and Nurr1 are active enhancers of SYN1 and SYN2 promoter activities. Other transcription factors have been recently identified to positively regulate SYN1 promoter activity, including Sp1 at sites immediately distal and proximal from the proximal CREB site (64), which raises the possibility that Sp1 and CREB might cooperate to control SYN1 expression.To conclude, we believe that soluble αSyn species are an intrinsic component of the sequence of events leading to dementia in AD, thereby exacerbating the severity of cognitive impairment, perhaps mediated by a selective lowering of synapsins. We also trust that these findings also apply to other synucleinopathies, in particular dementia with LBs. Although further studies are required to elucidate the mechanism governing the up-regulation of αSyn, this αSyn/synapsin axis might constitute an intriguing therapeutic target with the overarching goal to attenuate cognitive decline in patients at early stages of the disease.MethodsHuman Brain Tissue.Brain specimens and subject characteristics were described previously (65). The Religious Orders Study was approved by the Institutional Review Board of Rush University Medical Center and all participants gave informed consent, signed an Anatomical Gift Act for organ donation, and signed a repository consent to allow data and biospecimen sharing. The University of Minnesota Institutional Review Board approved this study.Transgenic Animals.Three Tg lines were used: (i) TgI2.2 mice expressing the WT form of human αSyn under the control of the mouse prion promoter (27), (ii) SNCA-null mice (66), and (iii) J20 mice (21). Bigenic J20×TgI2.2 mice resulted from the mating of TgI2.2 and J20 mice. All lines used were in the C57BL6 background strain. Both male and female animals were used in biochemical studies and BCM behavioral testing. All animal procedures and studies were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee and Institutional Review Board.Primary Cell Cultures.Mouse cortical cultures of neurons were prepared and used as described previously (67).Protein Extractions.Protocols for protein extractions are described previously (67).IC Delivery of Human αSyn Oligomers.Selected SEC fractions enriched or devoid in identified soluble αSyn species were coincubated for 30 min at 4 °C with 6 μL/μg of Chariot (Actif Motif). Mixtures were applied to the conditioned media of cells for 90 min at 37 °C.SEC.Protein separation was achieved as previously described (67).Western Blotting and Quantification.Experimental settings were described previously (18, 67).Spatial Reference Memory Assessments.Experiments were performed as described previously (18, 67).SI MethodsHuman Brain Tissue.Biochemical analyses were performed on human brain tissue from the (Brodman Area 20) of 84 subjects enrolled in the Religious Orders Study (68). The selection of the ITG for our analyses was guided by the following observations: (i) this region of the cerebral cortex shows reduced glucose utilization in AD and in asymptomatic individuals at risk genetically for AD (69); (ii) ITG gray matter thickness significantly predicts hippocampal volume loss in both amyloid+ and hyperphosphorylated tau+ individuals among MCI and AD individuals (70); and (iii) ITG amyloid loads and tangle density matched very well with average total brain amyloid burden (ρ = 0.946; P = 0.0001) and tangle density (ρ = 0.772; P = 0.0001). Cognitive status was assessed with 21 tests, 19 of which were summarized as a global measure of cognition and five cognitive domains (71). Selected cases were chosen to ensure that the three groups (NCI, MCI, and AD) diagnosed as previously described (71, 72) would not differ significantly from the whole Religious Orders Study cohort. Amyloid load and tangle density were quantified in six brain regions (73) and subjects further characterized by Braak stage, Consortium to Establish a Registry for Alzheimer’s Disease, and National Institute on Aging–Reagan pathologic diagnoses (74). The six brain regions included the hippocampus, entorhinal cortex, midfrontal gyrus, ITG, inferior parietal gyrus, and calcarine cortex, with averages determined by pooling amyloid load and tangle density from each area. The characteristics of the three clinical diagnostic groups are summarized elsewhere (18). The pathological characteristics of the clinical diagnostic groups selected for this study were similar to those of the entire Religious Orders Study cohort, whether assessed by amyloid load, tangle density, or LB density. Additional brain regions including the entorhinal cortex, the midfrontal gyrus (MF), the angular or inferior parietal gyrus (AG), and the Calcarine cortex (CALC) were used for comparative biochemical analyses with the ITG. The Religious Orders Study was approved by the Institutional Review Board of Rush University Medical Center and all participants gave informed consent, signed an Anatomical Gift Act for organ donation, and signed a repository consent to allow data and biospecimen sharing. The University of Minnesota Institutional Review Board approved this study.Transgenic Animals.WT and heterozygous Tg mice were used in this study. Tg mice expressing the WT form of human αSyn under the control of the mouse prion promoter (27), moPrp-HuSyn line I2-2 (WT), were used in conjunction with SNCA-null mice, which are deficient in endogenous mouse αSyn (66). In addition, Tg mice expressing the human form of APP with the Swedish (K670N, M671L) and Indiana (V717F) familial AD mutations directed by the platelet-derived growth factor chain promoter (21), APP line J20, were used. Bigenic J20×TgI2.2 mice were generated by crossing TgI2.2 and J20 mice. All lines used were in the C57BL6 background strain.Both male and female animals were used in biochemical studies and BCM behavioral testing. All animal procedures and studies were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee and Institutional Review Board.Protein Extractions.Soluble aggregation-prone protein levels in brain tissue were analyzed using the extraction protocol previously described (75, 76), with a detailed 32-step protocol explained in the latter. The goal of this lysis process is to fractionate proteins based on their cellular compartmentalization. The sequential separation allows the recovery of a predicted protein in its compartment of 75–90% (18, 67, 75, 76). Briefly, dissected frozen hemiforebrain tissues (125–200 mg) are gently dissociated in Nonidet P-40 lysis buffer [50 mM Tris⋅HCl (pH 7.6), 0.01% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 0.1% SDS] and centrifuged at 800 × g, to separate EC proteins contained in the supernatant. The remaining loose pellet is then lysed with TNT-lysis buffer [50 mM Tris⋅HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100], and centrifuged at 16,100 × g, to separate IC proteins present in the aqueous phase. The subsequent pellet is finally dissociated in RIPA-lysis buffer [50 mM Tris⋅HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 3% SDS, 1% deoxycholate], and centrifuged at 16,100 × g to separate membrane-bound proteins present in the supernatant. All supernatants were ultracentrifuged for 20 min at 100,000 × g. Before analysis, fractions were depleted of endogenous immunoglobulins by incubating lysates with 50 µL of Protein A-Sepharose, Fast Flow beads for 1 h at 4 °C, followed by 50 µL of Protein G-Sepharose, Fast Flow beads (GE Healthcare Life Sciences). Protein amounts were determined with the Bicinchoninic acid protein assay (BCA Protein Assay, Pierce).ELISA.IC-enriched protein fractions were used for the determination of o-αSyn levels in human brain tissue. Oligomeric forms of αSyn were identified using a custom sandwich ELISA, based on the principle documented earlier for Aβ. This ELISA used a capture antibody (LB509) with two detection antibodies conjugated to different infrared molecules: LB509-IR800 and A11-IR680. High-binding, clear bottom 96-well microplates (96 Well PS Microplate Flat Bottom without Lid, #655077, Greiner Bio-One) were treated with the capture antibody (1 μg per well) overnight, washed, and incubated with PBS containing 1% BSA before incubation with the brain lysate (250 ng per well). Following application of the detection antibody, the plates were read with the Odyssey system (Li-Cor Biosciences).Dot Blot.Two micrograms of EC-enriched or membrane-associated protein lysates were mixed with sterile filtered deionized water in a total volume of 2.5 μL. Each sample was then adsorbed onto a nitrocellulose membrane until dry. Following a brief activation in 10% methanol/TBS, the membrane was boiled in PBS to enhance antigen detection as previously described (76). Membranes were blocked in TBS containing 5% BSA for 60 min, then moved to the appropriate primary antibodies for overnight incubation at 4 °C. Following washes, anti-mouse IgG-IR800 (1:100,000) and anti-rabbit IgG-IR680 (1:150,000) secondary antibodies were used for detection with a Li-Cor Odyssey imager. All steps were performed without detergent to enhance A11/OC/F8H7/Syn33 binding of oligomeric species, as previously reported (75).Oligomer Disassembly.HFIP disassembly was performed on the EC and IC fractions of 4-, 7-, and 11-mo-old TgI2.2 brain extracts, along with recombinant αSyn. Concentrations of HFIP ranging from 0 to 100% were incubated with 2 mM EDTA and 50 µg of the brain extract or 0.5 µg of recombinant αSyn for 1 h at 37 °C, while agitated at 800 rpm. The solutions were vacuum-concentrated until dry, then reconstituted with loading buffer for gel electrophoresis.SEC.Protein extracts (250 μg) were loaded on Tricorn Superdex 75 columns (GE Healthcare Bio-Sciences Corp.) and run at a flow rate of 0.3 mL/min. Fractions of 250 μL of eluate in PBS with 0.1% Triton X-100, were collected using a BioLogic DuoFlow QuadTec 40 system (Bio-Rad) coupled to a microplate-format fraction collector. A280 was determined live during the experiments and confirmed following each run on a DTX800 Multimode microplate reader (Beckman Coulter).Western Blotting and Quantification.Electrophoresis was done using SDS/PAGE on precast 10–20% SDS-polyacrylamide Tris-Tricine gels, or 10.5–14% and 4–10.5% Tris⋅HCl gels (Bio-Rad). Protein levels were normalized by using 2–100 µg of protein per sample (depending on the targeted protein). The samples were resuspended with 4× Tricine loading buffer and boiled for 5 min before loading.Transfer.Proteins were transferred to 0.2-µm nitrocellulose membrane (Bio-Rad) following electrophoresis.Blotting.Membranes were blocked in TTBS (Tris-buffered saline-0.1%Tween20) containing 5% BSA (Sigma) for 1–2 h at room temperature, and probed with the appropriate antisera/antibodies diluted in 5% BSA-TTBS. Primary antibodies were probed with either anti-IgG immunoglobulins conjugated with biotin, HRP, or infrared dyes (Li-Cor Biosciences). When biotin-conjugated secondary antibodies were used, HRP- or infrared-conjugated Neutravidin (Pierce) or ExtrAvidin (Sigma) was added to amplify the signal. Blots were revealed on an Odyssey platform (Li-Cor Biosciences).Stripping.For reprobing, membranes were stripped using Restore Plus Stripping buffer (Pierce) for 5–180 min at room temperature, depending on the antibody affinity.Quantification.Densitometry analyses were performed using the Odyssey software (Li-Cor Biosciences). Each protein of interest was probed in three individual experiments under the same conditions. Quantification by software analysis, expressed as density light units, followed determination of experimental conditions ascertaining linearity in the detection of the signal. This method allows for a dynamic range of ∼100-fold above background. Respective averages were then determined across the triplicate Western blots. Normalization was performed against Actin or NeuN, which were also measured in triplicate.Please note that the color of the signal detected at 680 nm (red by default on the Li-Cor platform) was modified to magenta to allow colorblind individuals to distinguish both channels.Antibodies.The following primary antibodies were used in this study: LB509 (1:5,000–10,000), 4D6 (1:5,000), 4B12 (1:5,000), 6E10 (1:2,500; Covance and BioLegend), antisynapsin-I/II (1:1,000) (#106002), antisynapsin-III (1:1,000) (#106303), anticomplexin-1/2 (1:5,000; Synaptic Systems Inc), anti–β-Syn (1:1,000), anti-SYP (1:25,000), anti-NeuN (1:5,000), anti-CREB (1:2,000), and anti–pSer133-CREB (1:1,000; EMD Millipore), anti-Nurr1 (1:1,000; ThermoFisher Scientific and Santa Cruz Biotechnology), αSyn C-20 (1:1,000; Santa Cruz Biotechnology), rabbit-host antiactin (1:10,000; Sigma-Aldrich), mouse-host anti-actin (1:10,000; Pierce), Syn-33 (1:500), F8H7 (1:500), A11 (1:2,000), OC (1:2,000), and Officer (1:2,000; generated in-house by the R.K. laboratory).Transfections.HEK293T cells (ThermoFisher Scientific) were transiently transfected with the constructs indicated using the TransfastTransfection Reagent (Promega) as described by the manufacturer. For each transfection experiment, sister culture dishes were used to control the efficiency of transfection using an enhanced GFP-containing plasmid driven by a minimum promoter. Transfection efficiency corresponded to an average of ∼70% of transfected cells. The pRL-TK or pGL3-Control values provided by the Dual-Luciferase Reporter Assay System also served as internal control.Reporter Gene Assay.Two days after transfection, cells were lysed and luciferase activities were evaluated using the Dual Luciferase Reporter Assay System as described by the manufacturer (Promega). Values were normalized to the Firefly or Renilla luciferase activity depending on the reporter vector used. pGL3-SYN1m and pGL3-SYN1h constructs were provided by F.B. The reporters contain the −720/+15 and the −914/+11 fragments from mouse and human SYN1 promoters, respectively. The pEZX-PG02-hSYN2 vector containing the −1,203/+161 sequence of human SYN2 promoter and pEZX-PG02-Neg vectors were obtained from GeneCopoeia. The Nurr1 plasmid was a gift from Malin Parmar, Lund University, Lund, Sweden (Addgene plasmid # 35000). Treating cells with 10 µM forskolin (Tocris Bioscience) activated CREB as assessed by phosphorylation at serine 133 and nuclear translocation.Confocal Imaging.Triple or double-label immunofluorescence was performed as previously described (18) using Alexa Fluor-488, -555, -635–conjugated secondary antibodies (Molecular Probes, Invitrogen), treated for autofluorescence with 1% Sudan Black solution, and coverslipped with ProLong-DAPI mounting medium (Molecular Probes). Digital images were obtained using an Olympus IX81 FluoView1000 microscope. Raw image z-stacks were analyzed using Imaris7.x software suite (Bitplane Scientific Software).Barnes Circular Maze.The BCM (San Diego Instruments) was used for behavioral testing of WT and TgI2.2 transgenic mice. The apparatus consisted of an elevated circular platform (0.91-m diameter) with 20 holes (5-cm diameter) around the perimeter. One hole was connected to a dark escape recessed chamber, referred to as the target box. The maze was positioned in a room with large, simple visual cues attached on the surrounding walls. The protocol used was adapted from Sunyer et al. (77). Briefly, mice were habituated to the training room before each training day for 30 min in their cage. In addition, on the first day mice were placed at the center of the maze in a bottomless opaque cylinder for 60 s to familiarize the animals with the handling. Fifteen minutes later, training sessions started. Acquisition consisted of four trials per day for 4 d, separated by a 15-min intertrial interval. Each mouse was positioned in the center of the maze in an opaque cylinder, which was gently lifted and removed to start the session. The mice were allowed 180 s to find the target box on the first trial; all trials were 3 min long. At the end of the first 3 min, if the mouse failed to find the recessed escape box, it was gently guided to the chamber and allowed to stay in the target platform for 60 s. The location of the escape box was kept constant with respect to the visual cues, but the hole location of the target platform was changed randomly. An animal was considered to have found the escape chamber when its back legs crossed the horizontal plane of the platform. An animal was considered to have entered the escape chamber when its entire body was in the chamber and no longer visible on the platform. Retention was tested 24 h after the last training session (day 5) and 7 d after the initial probe (day 12). The same parameters were collected during the acquisition and retention phases using the ANY-maze software (San Diego Instruments, Stoelting Co.).Statistical Analyses.When variables were nonnormally distributed, nonparametric statistics were used (Spearman ρ correlation coefficients, Kruskal–Wallis nonparametric analysis of variance followed by Bonferroni-corrected two-group post hoc Mann–Whitney U tests). When variables were normally distributed, the following parametric statistics were used (one/two-way ANOVA followed by Bonferroni-corrected two-group post hoc Student t tests). Univariate repeated measures ANOVA were performed to determine the effects of day, transgene, and day × transgene interactions for behavioral experiments. Sample size was determined by power analysis to be able to detect statistically significant changes within a 20% variation of measured responses. Finally, none of the measured protein levels were normalized to our internal standards (actin, α-tubulin, βIII-tubulin, or NeuN) for the regression analyses performed. Analyses were performed using JMP 11 (SAS Institute).AcknowledgmentsWe thank Kenji Kanamura, Hoa Nguyen, and Chani Maher (Becker) for technical help, and the participants in the Religious Orders Study. This work was supported in part by NIH Grant R01AG044342, research Grant 4185-9227-14, and start-up funds from the University of Minnesota Foundation (to S.E.L.); and NIH Grants P30AG10161 and R01AG15819 (to D.A.B.).Footnotes↵1M.E.L. and S.J.G. contributed equally to this work.↵2To whom correspondence should be addressed. Email: lesne002{at}umn.edu.Author contributions: M.K.L., D.A.B., and S.E.L. designed research; M.E.L., S.J.G., F.A., M.L., G.B., M.A.S., H.S., C.M., and S.E.L. performed research; J.A.S., R.K., F.B., M.K.L., D.A.B., and S.E.L. contributed new reagents/analytic tools; M.E.L., S.J.G., F.A., M.L., G.B., M.A.S., H.S., C.M., J.A.S., R.K., F.B., D.A.B., and S.E.L. analyzed data; and M.E.L. and S.E.L. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1704698114/-/DCSupplemental. References↵Lasagna-Reeves CA, et al. (2011) Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice. Mol Neurodegener 6:39..OpenUrlCrossRefPubMed↵Larson ME, Lesné SE (2012) Soluble Aβ oligomer production and toxicity. 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Lesné Proceedings of the National Academy of Sciences Jun 2017, 114 (23) E4648-E4657; DOI: 10.1073/pnas.1704698114 α-Synuclein oligomers in Alzheimer’s disease Megan E. Larson, Susan J. Greimel, Fatou Amar, Michael LaCroix, Gabriel Boyle, Mathew A. Sherman, Hallie Schley, Camille Miel, Julie A. Schneider, Rakez Kayed, Fabio Benfenati, Michael K. Lee, David A. Bennett, Sylvain E. Lesné Proceedings of the National Academy of Sciences Jun 2017, 114 (23) E4648-E4657; DOI: 10.1073/pnas.1704698114 Sign up for the PNAS Highlights newsletter to get in-depth stories of science sent to your inbox twice a month: Relatively clean snow and ice in the Indus River Basin during the COVID-19 pandemic may have reduced meltwater in 2020, compared with the 20-year average. Atmospheric and climate conditions could have created a cloud greenhouse effect to warm Mars and support liquid surface water. Researchers report a safety guideline to limit airborne transmission of COVID-19 that goes beyond the six-foot social distancing guideline. Interventions include using rice husks, manipulating paddy water and soil, and genetic changes that could stop arsenic from reaching the grain. Going beyond conventional approaches, researchers are using carefully cultured bacterial communities to improve sewage treatment.