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In Alzheimer’s disease, amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However, there is limited information on how endogenous levels of tau, Aβ and other biomarkers relate to patient characteristics, or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures, we report that Aβ 1-40 and tau release levels vary with donor age and brain region, respectively. Release of other biomarkers such as KLK-6, NCAM-1, and Neurogranin vary between brain region, while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta, with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast, treatment with Aβ-containing Alzheimer’s disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue. Understanding synapse loss in Alzheimer’s disease has been hampered by a lack of human model systems. Here, the authors show that manipulation of physiological or pathological Aβ has differing effects on synapses in live human brain slice cultures.

Degeneration of synapses in Alzheimer’s disease (AD) strongly correlates with cognitive decline, and synaptic pathology contributes to disease pathophysiology. We recently observed that the strongest genetic risk factor for sporadic AD, apolipoprotein E epsilon 4 ( APOE 4), is associated with exacerbated synapse loss and synaptic accumulation of oligomeric amyloid beta in human AD brain. To begin to understand the molecular cascades involved in synapse loss in AD and how this is mediated by APOE , and to generate a resource of knowledge of changes in the synaptic proteome in AD, we conducted a proteomic screen and systematic in silico analysis of synaptoneurosome preparations from temporal and occipital cortices of human AD and control subjects with known APOE gene status. We examined brain tissue from 33 subjects (7–10 per group). We pooled tissue from all subjects in each group for unbiased proteomic analyses followed by validation with individual case samples. Our analysis identified over 5500 proteins in human synaptoneurosomes and highlighted disease, brain region, and APOE-associated changes in multiple molecular pathways including a decreased abundance in AD of proteins important for synaptic and mitochondrial function and an increased abundance of proteins involved in neuroimmune interactions and intracellular signaling.

The ability to learn progressively declines with age. Neural hyperactivity has been implicated in impairing cognitive plasticity with age, but the molecular mechanisms remain elusive. Here, we show that chronic excitation of the Caenorhabditis elegans O 2 -sensing neurons during ageing causes a rapid decline of experience-dependent plasticity in response to environmental O 2 concentration, whereas sustaining lower activity of O 2 -sensing neurons retains plasticity with age. We demonstrate that neural activity alters the ageing trajectory in the transcriptome of O 2 -sensing neurons, and our data suggest that high-activity neurons redirect resources from maintaining plasticity to sustaining continuous firing. Sustaining plasticity with age requires the K + -dependent Na + /Ca 2+ (NCKX) exchanger, whereas the decline of plasticity with age in high-activity neurons acts through calmodulin and the scaffold protein Kidins220. Our findings demonstrate directly that the activity of neurons alters neuronal homeostasis to govern the age-related decline of neural plasticity and throw light on the mechanisms involved.

Prion diseases or transmissible spongiform encephalopathies are characterized histopathologically by the accumulation of prion protein (PrP) ranging from diffuse deposits to amyloid plaques. Moreover, pathologic PrP isoforms (PrPSc) are detected by immunoblot analysis and used both as diagnostic markers of disease and as indicators of the presence of infectivity in tissues. It is not known which forms of PrP are associated with infectivity. To address this question, we performed bioassays using human brain extracts from two cases with phenotypically distinct forms of familial prion disease (Gerstmann-Sträussler-Scheinker P102L). Both cases had PrP accumulations in the brain, but each had different PrPSc isoforms. Only one of the brains had spongiform degeneration. Tissue from this case transmitted disease efficiently to transgenic mice (Tg PrP101LL), resulting in spongiform encephalopathy. In contrast, inoculation of tissue from the case with no spongiform degeneration resulted in almost complete absence of disease transmission but elicited striking PrP-amyloid deposition in several recipient mouse brains. Brains of these mice failed to transmit any neurological disease on passage, but PrP-amyloid deposition was again observed in the brains of recipient mice. These data suggest the possible isolation of an infectious agent that promotes PrP amyloidogenesis in the absence of a spongiform encephalopathy. Alternatively, the infectious agent may be rendered nonpathogenic by sequestration in amyloid plaques, or PrP amyloid can seed amyloid accumulation in the brain, causing a proteinopathy that is unrelated to prion disease. Formation of PrP amyloid may therefore not necessarily be a reliable marker of transmissible spongiform encephalopathy infectivity.

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP(Sc)) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP(Sc) propagation involves conversion from its normal isoform, PrP(C), by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP(Sen)) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP(Sc) (PrP(Res)). Scrapie brain dilutions up to 10(-8) and 10(-13) were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP(Res) levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP(C). Although the brains of 263K-affected mice had little immunoblot-detectable PrP(Res), RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP(Res) strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP(Res) levels. We also found that eQuIC, which incorporates a PrP(Sc) immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP(Sc).

Of all of the neuropathological changes observed in Alzheimer’s disease (AD), the loss of synapses correlates most strongly with cognitive decline. The precise mechanisms of synapse degeneration in AD remain unclear, although strong evidence indicates that pathological forms of both amyloid beta and tau contribute to synaptic dysfunction and loss. Synaptic mitochondria play a potentially important role in synapse degeneration in AD. Many studies in model systems indicate that amyloid beta and tau both impair mitochondrial function and impair transport of mitochondria to synapses. To date, much less is known about whether synaptic mitochondria are affected in human AD brain. Here, we used transmission electron microscopy to examine synapses and synaptic mitochondria in two cortical regions (BA41/42 and BA46) from eight AD and nine control cases. In this study, we observed 3000 synapses and find region-specific differences in synaptic mitochondria in AD cases compared to controls. In BA41/42, we observe a fourfold reduction in the proportion of presynaptic terminals that contain multiple mitochondria profiles in AD. We also observe ultrastructural changes including abnormal mitochondrial morphology, the presence of multivesicular bodies in synapses, and reduced synapse apposition length near plaques in AD. Together, our data show region-specific changes in synaptic mitochondria in AD and support the idea that the transport of mitochondria to presynaptic terminals or synaptic mitochondrial dynamics may be altered in AD.

Background There is currently a need for an imaging biomarker of synaptic density that would allow the tracking of changes associated with neurodegeneration, disease progression and possible therapeutic response. The aim of this study was to use homogenate binding techniques to assess [3H]SynVesT‐1 binding to synaptic vesicle glycoprotein 2A (SV2A), found in pre‐synaptic terminals, to understand its potential as a synaptic density biomarker. Method Postmortem brain tissues from 70 subjects, across 3 different regions (entorhinal cortex (EC), cerebellum and centrum semiovale) and two homogenate fractions (total homogenate (TH) and synaptoneurosome (SN)) were used to determine the binding parameters (binding affinity (KD), and maximal binding sites (BMAX)) of [3H]SynVesT‐1. Sex‐matched subjects were initially divided into 3 groups: control (Braak Stage 0‐II), early Alzheimer's Disease (E‐AD) (Braak Stage III‐IV) and late AD (L‐AD) (Braak Stage V‐VI). Result A saturable specific binding signal was observed in EC and cerebellum homogenates from all three groups. A suitable binding curve could not be applied to the centrum semiovale, with less specific binding, and greater variability across cases. The KD of [3H]SynVesT‐1 was comparable across groups and homogenate preparations (TH and SN), ranging from 3‐7 nM. In initial groupwise analyses, the BMAX ranged from 2278 ± 101 fmol/mg protein in L‐AD, to 2788 ± 109 fmol/mg protein in control in the TH preparations from the EC. SN homogenates for the EC show BMAX ranged from 3958 ± 183 fmol/mg protein in L‐AD, to 5193 ± 316 fmol/mg protein in E‐AD. In cerebellum TH, BMAX ranged from 2263 ± 88 fmol/mg protein in control, to 2681 ± 186 fmol/mg protein in E‐AD. In cerebellum SN homogenates, BMAX ranged from 2790± 134 fmol/mg protein in L‐AD, to 4085 ± 239 fmol/mg protein in E‐AD. Conclusion Initial results show group‐wise differences in the BMAX of [3H]SynVesT‐1 in EC, with E‐AD subjects showing greater binding signal. This may suggest compensatory mechanisms occurring early in the progression of the disease. This is part of the SV2A PET Project, a program of the FNIH Biomarker Consortium.

Background Numerous studies have used Synaptic Vesicle Glycoprotein 2A (SV2A) as a radioligand target during in‐vivo Positron Emission Tomography (PET) in humans to investigate the synaptic loss that strongly correlates with cognitive decline in Alzheimer's disease (AD). These studies showed a decrease in the SV2A PET signal in AD patient brains compared to controls highlighting the signals potential as a clinical biomarker. However, it is currently unclear whether this decreasing signal reflects synapse loss or a loss of SV2A from within the remaining synapses. This study uses array tomography, a high‐resolution imaging technique allowing quantification of synapse density and protein colocalisation, with the intent to validate whether SV2A could be used to accurately detect synapse loss in AD. Method We compare SV2A puncta density, localisation and intensity with synaptophysin, a well characterised pre‐synaptic marker previously used to identify AD‐associated synapse loss, in human post‐mortem tissue samples from the inferior temporal gyrus, dorsolateral prefrontal cortex, entorhinal cortex, and cerebellum from end‐stage AD and age‐matched control subjects (n = 11‐19 cases per group for each brain region). Data are analysed with linear mixed effects models and Spearman's correlations. Result We observe strong correlations between SV2A puncta density and synaptophysin puncta density within the same tissue samples, indicating SV2A is a reliable marker of synapse density. We further observe differences between brain regions in synapse density and in disease‐associated synapse changes. Preliminary analyses of SV2A intensity within synaptic puncta do not show differences between AD and control across regions indicating remaining synapses likely do not have altered levels of SV2A protein. Conclusion These data indicate that changes in SV2A signal observed in AD with PET imaging likely reflect changes in synapse density rather than changes in protein levels within remaining synapses. Future work with the Foundations for the National Institutes of Health SV2A project team will integrate these data with in vivo PET imaging and molecular analyses of post‐mortem tissue to provide further understanding of the biological underpinnings of SV2A PET signal with the goal of determining whether SV2A PET will be a reliable marker of synapse density for use in future clinical use.

Numerous studies have used Synaptic Vesicle Glycoprotein 2A (SV2A) as a radioligand target during in-vivo Positron Emission Tomography (PET) in humans to investigate the synaptic loss that strongly correlates with cognitive decline in Alzheimer's disease (AD). These studies showed a decrease in the SV2A PET signal in AD patient brains compared to controls highlighting the signals potential as a clinical biomarker. However, it is currently unclear whether this decreasing signal reflects synapse loss or a loss of SV2A from within the remaining synapses. This study uses array tomography, a high-resolution imaging technique allowing quantification of synapse density and protein colocalisation, with the intent to validate whether SV2A could be used to accurately detect synapse loss in AD. We compare SV2A puncta density, localisation and intensity with synaptophysin, a well characterised pre-synaptic marker previously used to identify AD-associated synapse loss, in human post-mortem tissue samples from the inferior temporal gyrus, dorsolateral prefrontal cortex, entorhinal cortex, and cerebellum from end-stage AD and age-matched control subjects (n = 11-19 cases per group for each brain region). Data are analysed with linear mixed effects models and Spearman's correlations. We observe strong correlations between SV2A puncta density and synaptophysin puncta density within the same tissue samples, indicating SV2A is a reliable marker of synapse density. We further observe differences between brain regions in synapse density and in disease-associated synapse changes. Preliminary analyses of SV2A intensity within synaptic puncta do not show differences between AD and control across regions indicating remaining synapses likely do not have altered levels of SV2A protein. These data indicate that changes in SV2A signal observed in AD with PET imaging likely reflect changes in synapse density rather than changes in protein levels within remaining synapses. Future work with the Foundations for the National Institutes of Health SV2A project team will integrate these data with in vivo PET imaging and molecular analyses of post-mortem tissue to provide further understanding of the biological underpinnings of SV2A PET signal with the goal of determining whether SV2A PET will be a reliable marker of synapse density for use in future clinical use.

Synapse loss correlates with cognitive decline in Alzheimer’s disease, and soluble oligomeric amyloid beta (Aβ) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aβ leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aβ and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of Aβ binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Förster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD ( n  = 11) and control cases ( n  = 9). Within presynapses and post-synaptic densities, oligomeric Aβ generates a FRET signal with transmembrane protein 97. Further, Aβ generates a FRET signal with cellular prion protein, and post-synaptic density 95 within post synapses. Transmembrane protein 97 is also present in a higher proportion of post synapses in Alzheimer’s brain compared to controls. We inhibited Aβ/transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the allosteric modulator CT1812. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and Aβ. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aβ when neurons are challenged with human Alzheimer’s brain homogenate. Transcriptional changes are induced by Aβ including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of Aβ in human Alzheimer’s disease brain where it may mediate synaptotoxicity.