Human Alzheimer's Disease Tissue Research Articles

Label-Free In Situ Chemical Characterization of Amyloid Plaques in Human Brain Tissues.

The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer's disease. Altered metabolism of essential biometals is another feature of Alzheimer's, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer's disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C-H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer's brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer's disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer's disease.

Mass spectrometry imaging highlights dynamic patterns of lipid co-expression with Aβ plaques in mouse and human brains.

Lipids play crucial roles in the susceptibility and brain cellular responses to Alzheimer's disease (AD) and are increasingly considered potential soluble biomarkers in cerebrospinal fluid (CSF) and plasma. To delineate the pathological correlations of distinct lipid species, we conducted a comprehensive characterization of both spatially localized and global differences in brain lipid composition in AppNL-G-F mice with spatial and bulk mass spectrometry lipidomic profiling, using human amyloid-expressing (h-Aβ) and WT mouse brains controls. We observed age-dependent increases in lysophospholipids, bis(monoacylglycerol) phosphates, and phosphatidylglycerols around Aβ plaques in AppNL-G-F mice. Immunohistology-based co-localization identified associations between focal pro-inflammatory lipids, glial activation, and autophagic flux disruption. Likewise, in human donors with varying Braak stages, similar studies of cortical sections revealed co-expression of lysophospholipids and ceramides around Aβ plaques in AD (Braak stage V/VI) but not in earlier Braak stage controls. Our findings in mice provide evidence of temporally and spatially heterogeneous differences in lipid composition as local and global Aβ-related pathologies evolve. Observing similar lipidomic changes associated with pathological Aβ plaques in human AD tissue provides a foundation for understanding differences in CSF lipids with reported clinical stage or disease severity.

Spatial Neurolipidomics at the Single Amyloid-β Plaque Level in Postmortem Human Alzheimer's Disease Brain.

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Amyloid Precursor Protein 𝛽CTF accumulates in synapses in sporadic and genetic forms of Alzheimer's disease.

Amyloid Precursor Protein (APP) 𝛽-C-terminal fragment (𝛽CTF) may have a neurotoxic role in Alzheimer´s disease (AD). 𝛽CTF accumulates in the brains of patients with sporadic (SAD) and genetic forms of Alzheimer's Disease (AD). Synapses degenerate early during the pathogenesis of AD. We studied whether the 𝛽CTF accumulates in synapses in SAD, autosomal dominant AD (ADAD) and Down syndrome (DS). We used Array tomography to determine APP at synapses in human AD tissue. We measured 𝛽CTF, A𝛽40, A𝛽42 and phosphorylated tau181 (p-tau181) concentrations in brain homogenates and synaptosomes of frontal and temporal cortex of SAD, ADAD, DS and controls. APP colocalized with pre- and post-synaptic markers in human AD brains. APP 𝛽CTF was enriched in AD synaptosomes. We demonstrate that 𝛽CTF accumulates in synapses in SAD, ADAD and DS. This finding might suggest a role for 𝛽CTF in synapse degeneration. Therapies aimed at mitigating 𝛽CTF accumulation could be potentially beneficial in AD.

Absence of microglia promotes diverse pathologies and early lethality in Alzheimer's disease mice.

SUMMARYMicroglia are strongly implicated in the development and progression of Alzheimer’s disease (AD), yet their impact on pathology and lifespan remains unclear. Here we utilize a CSF1R hypomorphic mouse to generate a model of AD that genetically lacks microglia. The resulting microglial-deficient mice exhibit a profound shift from parenchymal amyloid plaques to cerebral amyloid angiopathy (CAA), which is accompanied by numerous transcriptional changes, greatly increased brain calcification and hemorrhages, and premature lethality. Remarkably, a single injection of wild-type microglia into adult mice repopulates the microglial niche and prevents each of these pathological changes. Taken together, these results indicate the protective functions of microglia in reducing CAA, blood-brain barrier dysfunction, and brain calcification. To further understand the clinical implications of these findings, human AD tissue and iPSC-microglia were examined, providing evidence that microglia phagocytose calcium crystals, and this process is impaired by loss of the AD risk gene, TREM2.

Impact of Sterilization Methods on the Seeding Ability of Human Tau Proteopathic Seeds.

The protein tau, when misfolded in neurodegenerative diseases, has several prion-like properties including being able to spread by cell-to-cell transfer, induce templated seeding, and exist in distinct conformational strains. These properties of transmission may present health hazards when lesion-containing biospecimens are used in research and neuropathology laboratories. We evaluated the impact standard sterilization and cleaning methods have on the capacity of tau seeds to induce aggregation. We employed a previously developed, highly sensitive FRET-based biosensor assay to assess remnant tau seeding after exposure to these procedures. For tau species derived from human Alzheimer disease tissue (brain homogenate and sarkosyl-insoluble fibrils), both autoclaving and incubation in 90.6% formic acid were sufficient to reduce tau bioactivity. By contrast, boiling was not always effective in completely blocking bioactivity in the seeding assay. Notably, only formic acid incubation was able to produce a similar reduction in tissue from a P301L mutant tau mouse model of tauopathy. Our study highlights nuances in methods for inactivation of tau seeding which may support adapted tissue processing procedures, especially in research settings. These findings also highlight the importance of universal precautions when handling human neuropathological and research laboratory materials.

Alzheimer amyloid-β- peptide disrupts membrane localization of glucose transporter 1 in astrocytes: implications for glucose levels in brain and blood

Alzheimer's disease (AD) is associated with disturbances in blood glucose regulation, and type-2 diabetes elevates the risk for dementia. A role for amyloid-β peptide (Aβ) in linking these age-related conditions has been proposed, tested primarily in transgenic mouse lines that overexpress mutated amyloid precursor protein (APP). Because APP has its own impacts on glucose regulation, we examined the BRI-Aβ42 line (“Aβ42-tg”), which produces extracellular Aβ1-42 in the CNS without elevation of APP. We also looked for interactions with diet-induced obesity (DIO) resulting from a high-fat, high-sucrose (“western”) diet. Aβ42-tg mice were impaired in both spatial memory and glucose tolerance. Although DIO induced insulin resistance, Aβ1-42 accumulation did not, and the impacts of DIO and Aβ on glucose tolerance were merely additive. Aβ42-tg mice exhibited no significant differences from wild-type in insulin production, body weight, lipidemia, appetite, physical activity, respiratory quotient, an-/orexigenic factors, or inflammatory factors. These negative findings suggested that the phenotype in these mice arose from perturbation of glucose excursion in an insulin-independent tissue. To wit, cerebral cortex of Aβ42-tg mice had reduced glucose utilization, similar to human patients with AD. This was associated with insufficient trafficking of glucose transporter 1 to the plasma membrane in parenchymal brain cells, a finding also documented in human AD tissue. Together, the lower cerebral metabolic rate of glucose and diminished function of parenchymal glucose transporter 1 indicate that aberrant regulation of blood glucose in AD likely reflects a central phenomenon, resulting from the effects of Aβ on cerebral parenchyma, rather than a generalized disruption of hypothalamic or peripheral endocrinology. The involvement of a specific glucose transporter in this deficit provides a new target for the design of AD therapies.

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin.

Deposition of amyloid beta protein (Aβ) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer's disease (AD). Therefore, detection of the presence of Aβ in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and Aβ-specific antibodies have been used to detect Aβ histochemically in AD brain tissue. Use of these compounds for Aβ detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for Aβ, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of Aβ plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label Aβ plaques from both a genetic mouse model of 5x familial Alzheimer's disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as Aβ-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image Aβ plaques when compared to these conventional dyes and is comparable to Aβ-specific antibodies. In addition, Cur binds with most Aβ species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for Aβ plaques.

Neuronal calcineurin transcriptional targets parallel changes observed in Alzheimer disease brain.

Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Read the Editorial Highlight for this article on page8.

Immune hyperreactivity of Aβ plaque-associated microglia in Alzheimer's disease

Alzheimer's disease (AD) is strongly associated with microglia-induced neuroinflammation. Particularly, Aβ plaque-associated microglia take on an “activated” morphology. However, the function and phenotype of these Aβ plaque-associated microglia are not well understood. We show hyperreactivity of Aβ plaque-associated microglia upon systemic inflammation in transgenic AD mouse models (i.e., 5XFAD and APP23). Gene expression profiling of Aβ plaque-associated microglia (major histocompatibility complex II+ microglia) isolated from 5XFAD mice revealed a proinflammatory phenotype. The upregulated genes involved in the biological processes (gene ontology terms) included: “immune response to external stimulus” such as Axl, Cd63, Egr2, and Lgals3, “cell motility”, such as Ccl3, Ccl4, Cxcr4, and Sdc3, “cell differentiation”, and “system development”, such as St14, Trpm1, and Spp1. In human AD tissue with similar Braak stages, expression of phagocytic markers and AD-associated genes, including HLA-DRA, APOE, AXL, TREM2, and TYROBP, was higher in laser-captured early-onset AD (EOAD) plaques than in late-onset AD plaques. Interestingly, the nonplaque parenchyma of both EOAD and late-onset AD brains, the expression of above-mentioned markers were similarly low. Here, we provide evidence that Aβ plaque-associated microglia are hyperreactive in their immune response and phagocytosis in the transgenic AD mice as well as in EOAD brain tissue. We suggest that Aβ plaque-associated microglia are the primary source of neuroinflammation related to AD pathology.

Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer's disease.

BackgroundThe chemokine interleukin-8 (IL-8) and its receptor CXCR2 contribute to chemotactic responses in Alzheimer’s disease (AD); however, properties of the ligand and receptor have not been characterized in animal models of disease. The primary aim of our study was to examine effects of pharmacological antagonism of CXCR2 as a strategy to inhibit receptor-mediated inflammatory reactivity and enhance neuronal viability in animals receiving intrahippocampal injection of amyloid-beta (Aβ1–42).MethodsIn vivo studies used an animal model of Alzheimer’s disease incorporating injection of full-length Aβ1–42 into rat hippocampus. Immunohistochemical staining of rat brain was used to measure microgliosis, astrogliosis, neuronal viability, and oxidative stress. Western blot and Reverse Transcription PCR (RT-PCR) were used to determine levels of CXCR2 in animal tissue with the latter also used to determine expression of pro-inflammatory mediators. Immunostaining of human AD and non-demented (ND) tissue was also undertaken.ResultsWe initially determined that in the human brain, AD relative to ND tissue exhibited marked increases in expression of CXCR2 with cell-specific receptor expression prominent in microglia. In Aβ1–42-injected rat brain, CXCR2 and IL-8 showed time-dependent increases in expression, concomitant with enhanced gliosis, relative to controls phosphate-buffered saline (PBS) or reverse peptide Aβ42–1 injection. Administration of the competitive CXCR2 antagonist SB332235 to peptide-injected rats significantly reduced expression of CXCR2 and microgliosis, with astrogliosis unchanged. Double staining studies demonstrated localization of CXCR2 and microglial immunoreactivity nearby deposits of Aβ1–42 with SB332235 effective in inhibiting receptor expression and microgliosis. The numbers of neurons in granule cell layer (GCL) were reduced in rats receiving Aβ1–42, compared with PBS, with administration of SB332235 to peptide-injected animals conferring neuroprotection. Oxidative stress was indicated in the animal model since both 4-hydroxynonenal (4-HNE) and hydroethidine (HEt) were markedly elevated in Aβ1–42 vs PBS-injected rat brain and diminished with SB332235 treatment.ConclusionOverall, the findings suggest critical roles for CXCR2-dependent inflammatory responses in an AD animal model with pharmacological modulation of the receptor effective in inhibiting inflammatory reactivity and conferring neuroprotection against oxidative damage.

Do current therapeutic anti-Aβ antibodies for Alzheimer's disease engage the target?

Reducing amyloid-β peptide (Aβ) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aβ is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aβ in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aβ with high affinity. All of the antibodies were able to bind Aβ in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety ofN-terminally truncated Aβ species, the Aβ detected using solanezumab was barely above detection limits while crenezumab did not detect any Aβ. None of the antibodies were able to detect any Aβ species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aβ related proteins. Bapineuzumab demonstrated target engagement with brain Aβ, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.

Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease

In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum–lacunosum–moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame–shifted isoform, GFAP+1, staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP+1 isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP+1 isoforms in astrocytes and their possible role in AD pathology.

Reactive glia show increased immunoproteasome activity in Alzheimer’s disease

The proteasome is the major protein degradation system within the cell, comprised of different proteolytic subunits; amyloid-β is thought to impair its activity in Alzheimer's disease. Neuroinflammation is a prominent hallmark of Alzheimer's disease, which may implicate an activation of the immunoproteasome, a specific proteasome variant induced by immune signalling that holds slightly different proteolytic properties than the constitutive proteasome. Using a novel cell-permeable proteasome activity probe, we found that amyloid-β enhances proteasome activity in glial and neuronal cultures. Additionally, using a subunit-specific proteasome activity assay we showed that in the cortex of the APPswePS1dE9 plaque pathology mouse model, immunoproteasome activities were strongly increased together with increased messenger RNA and protein expression in reactive glia surrounding plaques. Importantly, this elevated activity was confirmed in human post-mortem tissue from donors with Alzheimer's disease. These findings are in contrast with earlier studies, which reported impairment of proteasome activity in human Alzheimer's disease tissue and mouse models. Targeting the increased immunoproteasome activity with a specific inhibitor resulted in a decreased expression of inflammatory markers in ex vivo microglia. This may serve as a potential novel approach to modulate sustained neuroinflammation and glial dysfunction associated with Alzheimer's disease.

Molecule of the month: miRNA and proteins in Alzheimer's disease

The analysis of Alzheimer's disease is an important topic of research because the results and findings help elucidate problems associated with aging as well as other diseases including Downs' syndrome, Parkinson's disease, dementia with Lewy bodies, Multiple Sclerosis, Amyotrophic Lateral Sclerosis,and infectious disease including NeuroAIDS. Here we review just a few points from an ever growing and productive literature. A recent article dealt with oxidation effects and neurodegeneration [1]. The hypothesis addressed was that oxidation of RNA affects miRNA and protein expression. In human as well as in rodent brain, neurodegeneration (neuronal) that is age-associated was demonstrated that is consequent to oxidation of nucleic acids and proteins. Such neuronal degenerative factors have also been analyzed in dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Parkinson disease, and Alzheimer disease. These findings occurred in cellular as well as animal models. Coding RNA as well as non-coding RNA were affected and deleterious effects on cell fate were analyzed. Such studies are needed for developing strategies of therapy. A recent study also analyzed miRNAs in Alzheimer's disease pathogenesis [2]. The analysis of extracellular fluid from neocortex associated with temporal lobe from patients who had Alzheimer's disease indicated increased miRNAs. These miRNAs included miRNA-155 and miRNA-146a that are proinflammatory. Moreover, when cell culture medium was used with these miRNA added, Alzheimer's disease-like gene expression occurred. This profile was associated with downregulation of complement factor H (CFH), a repressor of the innate immune response. Anti-miRNA approaches reduced the pro-inflammatory effects. Additional miRNAs, miRNA-9 and miRNA-125bwere involved as well [3]. These miRNAs were regulated by NF-кB andbound independently to the mRNA 3'- untranslated region to reduce CFH translation. The authors of an article in 2011 [4] stated that transcription factor, TAp73, drove expression of miRNA-34a (but not miRNA-34b or miRNA-34c). This was associated with decreased expression of synaptic proteins synaptotagmin-1and syntaxin-1A. These controlled molecular reactions generally occurred in retinoid-stimulated differentiation of neuroblastoma cells and rodents. The synaptotagmin-1 protein expression changes also occurred in Alzheimer's disease brain tissue. These observations were extended to non-classical infectious disease related to prion infections. Several neurotrophic RNA and DNA viruses induced miRNA-146a in human brain cells. In addition, oxidative stress, neurotoxic metals,pro-inflammatory cytokines,as well as Aβ42 peptideupregulated miRNA-146a as wellin primary cell human glial-neuronal cocultures. These findings were in Alzheimer's disease and murine scrapie studies. miRNA-146a quantitieswere related to disease severity and inflammatory neuropathology in Alzheimer's disease. Moreover, findings were similar in Gerstmann-Straussler- Scheinker syndrome and sporadicCreutzfeldt-Jakob disease [5]. In addition, Aβ oligopeptide down regulated miRNA-9 and miRNA-181c in earlier studies. This occurred in hippocampal cultures. In addition, APP23 transgenicmice and human Alzheimer's disease tissue showed deposition of Aβ. Moreover, APP is a target of miRNA regulation and additional 3'- untranslated regions were identified from several mRNAs. These are down regulatedbymiR-9 and -181c, separately and contemporaneously and includedTGFBI, TRIM2, SIRT1 and BTBD3 [6]. NF-kB has a very large well-known number of protein-protein interactions and is not included here. Based on the literature, the protein Tap73 is more often recognized as TP73. Tap73 was absent in the GNCPro database and so the term TP73 was used. The figure illustrates various gene interactions among the proteins mentioned above:complement factor H (CFH), synaptotagmin-1 (SYT1), syntaxin-1A (STX1A), tumor protein p73 transcription factor (TP73), transforming growth factor (TGFBI), tripartite motif containing 2(TRIM2),sirtuin 1(SIRT1), andBTB (POZ) domain containing 3(BTBD3). It is left as a puzzle for the interested reader to identify the various genes and their functions in the Figure 1 [7–9]. Figure 1 Network of input proteins (complement factor H, synaptotagmin-1, syntaxin-1A, TP73, TGFBI, TRIM2, SIRT1, andBTBD3) and the input neighbors of these proteins. In this figure, line-colors and various interactions with other genes are red Down-regulation, ...

Neurites containing the neurofilament-triplet proteins are selectively vulnerable to cytoskeletal pathology in Alzheimer’s disease and transgenic mouse models

Amyloid-β plaque accumulation in Alzheimer’s disease (AD) is associated with dystrophic neurite (DN) formation and synapse loss in principal neurons, but interneuron pathology is less clearly characterized. We compared the responses of neuronal processes immunoreactive for either neurofilament triplet (NF+) or calretinin (CR+) to fibrillar amyloid (Aβ) plaques in human end-stage and preclinical AD, as well as in APP/PS1 and Tg2576 transgenic mouse AD models. Neurites traversing the Aβ plaque core, edge, or periphery, defined as 50, 100, and 150% of the plaque diameter, respectively, in human AD and transgenic mouse tissue were compared to age-matched human and wild-type mouse controls. The proportion of NF+ neurites exhibiting dystrophic morphology (DN) was significantly larger than the proportion of dystrophic CR+ neurites in both human AD and transgenic mice (p < 0.01). Additionally, the number of NF+, but not CR+, DNs, correlated with Aβ plaque size. We conclude that CR+ interneurons appear to be more resistant than NF+ neurons to AD-mediated cytoskeletal pathology.

Tau Accumulation Causes Mitochondrial Distribution Deficits in Neurons in a Mouse Model of Tauopathy and in Human Alzheimer's Disease Brain

Neurofibrillary tangles (NFT), intracellular inclusions of abnormal fibrillar forms of microtubule associated protein tau, accumulate in Alzheimer's disease (AD) and other tauopathies and are believed to cause neuronal dysfunction, but the mechanism of tau-mediated toxicity are uncertain. Tau overexpression in cell culture impairs localization and trafficking of organelles. Here we tested the hypothesis that, in the intact brain, changes in mitochondrial distribution occur secondary to pathological changes in tau. Array tomography, a high-resolution imaging technique, was used to examine mitochondria in the reversible transgenic (rTg)4510, a regulatable transgenic, mouse model and AD brain tissue. Mitochondrial distribution is progressively disrupted with age in rTg4510 brain, particularly in somata and neurites containing Alz50-positive tau aggregates. Suppression of soluble tau expression with doxycycline resulted in complete recovery of mitochondrial distribution, despite the continued presence of aggregated tau. The effect on mitochondrial distribution occurs without concomitant alterations in neuropil mitochondrial size, as assessed by both array tomography and electron microscopy. Similar mitochondrial localization alterations were also observed in human AD tissue in Alz50+ neurons, confirming the relevance of tau to mitochondrial trafficking observed in this animal model. Because abnormalities reverted to normal if soluble tau was suppressed in rTg4510 mice, even in the continued presence of fibrillar tau inclusions, we suggest that soluble tau plays an important role in mitochondrial abnormalities, which likely contribute to neuronal dysfunction in AD.

Target Gene Repression Mediated by miRNAs miR-181c and miR-9 Both of Which Are Down-regulated by Amyloid-β

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that are essential for normal brain development and function. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer's disease (AD) that is characterized by amyloid-β (Aβ) and tau deposition in brain. How deregulated miRNAs contribute to AD is not understood, as their dysfunction could be both a cause and a consequence of disease. To address this question we had previously profiled miRNAs in models of AD. This identified miR-9 and -181c as being down-regulated by Aβ in hippocampal cultures. Interestingly, there was a remarkable overlap with those miRNAs that are deregulated in Aβ-depositing APP23 transgenic mice and in human AD tissue. While the Aβ precursor protein APP itself is a target of miRNA regulation, the challenge resides in identifying further targets. Here, we expand the repertoire of miRNA target genes by identifying the 3' untranslated regions (3' UTRs) of TGFBI, TRIM2, SIRT1 and BTBD3 as being repressed by miR-9 and -181c, either alone or in combination. Taken together, our study identifies putative target genes of miRNAs miR-9 and 181c, which may function in brain homeostasis and disease pathogenesis.

Specific recognition of the C-terminal end of Aβ 42 by a high affinity monoclonal antibody

The neurotoxic peptide Aβ 42 is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of Aβ 42 with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of Aβ 42 including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of Aβ. This has been accomplished by using a unique antigen construct made by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of Aβ 42. The generated Mab Aβ1.1 is indeed specific for the C-terminal end of Aβ 42 to which it binds with high affinity. Mab Aβ1.1 recognizes the epitope in human AD tissue and stains plaques with high specificity. Therefore the monoclonal antibody can thus be useful in the histological investigations of the AD pathology.

Aberrant Expression of Myeloperoxidase in Astrocytes Promotes Phospholipid Oxidation and Memory Deficits in a Mouse Model of Alzheimer Disease

Myeloperoxidase (MPO) is expressed in Alzheimer disease (AD) but not normal aged brain. A functional -463G/A MPO promoter polymorphism has been associated with AD risk through as yet unidentified mechanisms. Here we report that human MPO-463G allele, but not MPO-463A or mouse MPO, is strongly expressed in astrocytes and deposited in plaques in huMPO transgenic mice crossed to the APP23 model. MPO is similarly expressed in astrocytes in human AD tissue. In cortical homogenates of the MPOG-APP23 model, MPO expression correlated with increased levels of a lipid peroxidation product, 4-hydroxynonenal. Fluorescence high-performance liquid chromatography and electrospray ionization mass spectroscopy identified selective accumulation of phospholipid hydroperoxides in two classes of anionic phospholipids, phosphatidylserine (PS-OOH) and phosphatidylinositol (PI-OOH). The same molecular species of PS-OOH and PI-OOH were elevated in human AD brains as compared with non-demented controls. Augmented lipid peroxidation in MPOG-APP23 mice correlated with greater memory deficits. We suggest that aberrant huMPO expression in astrocytes leads to a specific pattern of phospholipid peroxidation and neuronal dysfunction contributing to AD.