Search

Alzheimer’s disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid β-protein (Aβ42) forms and novel intraneuronal Aβ oligomers (AβOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aβ uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aβ42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aβ pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aβ42, far-peripheral AβOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Alzheimer’s disease (AD) pathologies were discovered in the easily accessible neurosensory retina. Yet, their specific nature, topographical distribution, and relationship with disease status remain undefined. Here, we histologically determined burden and spatial distribution of amyloid β-protein (Aβ42), intraneuronal scFvA13+-Aβ species, macro- and microgliosis, and atrophy in superior- and inferior-temporal retinas of human donors with mild cognitive impairment (MCI) or AD versus normal cognition. AD and MCI patients had enhanced retinopathy, predominantly affecting inner layers and peripheral subregions, which quantitatively correlated with severity of cerebral amyloid, tau, and neurodegeneration, and cognitive scores. In advanced clinical stages AD retinopathy further affected central outer segments. Increased retinal macrogliosis and Aβ-phagocytosing microglia were detected in MCI and AD patients. Further, distinct proteome profiles of AD retinas were identified, displaying greater overlap with the temporal cortices than with hippocampi or cerebella. AD retinas exhibited upregulated inflammatory and neurodegenerative processes and downregulated oxidative-phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps AD retinopathy, demonstrating the quantitative relationship with brain pathology and cognition.

Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, phagocytic, pro-healing phenotype, whereas OPN inhibition triggered a pro-inflammatory phenotype. Here, we applied a global proteome profiling via mass spectrometry analysis to gain a mechanistic understanding of OPN suppression versus induction in macrophages. We identified over 630 differentially expressed proteins (DEPs) in OPN knockout (OPNKO) or GA-stimulated versus wild type (WT) macrophages. Two topmost downregulated DEPs in OPN-deficient macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We confirmed UCHL1 expression in BMMΦ, which was previously recognized as a neuronal-specific protein. Further, immunoprecipitation assays suggest a direct interaction of OPN and UCHL1 proteins. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (ROS, Lamp1/2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, Western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis (proteostasis) in macrophages—inducing apoptosis and inhibiting translation—whereas GA-stimulated OPN induction restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostasis via regulation of cell viability, UCHL1-UPS, and protein synthesis, indicating its potential application in immunotherapy.

Alzheimer’s disease (AD) is a major risk for the aging population. The pathological hallmarks of AD—an abnormal deposition of amyloid β-protein (Aβ) and phosphorylated tau (pTau)—have been demonstrated in the retinas of AD patients, including in prodromal patients with mild cognitive impairment (MCI). Aβ pathology, especially the accumulation of the amyloidogenic 42-residue long alloform (Aβ42), is considered an early and specific sign of AD, and together with tauopathy, confirms AD diagnosis. To visualize retinal Aβ and pTau, state-of-the-art methods use fluorescence. However, administering contrast agents complicates the imaging procedure. To address this problem, we developed a label-free hyperspectral imaging method to detect the spectral signatures of Aβ42 and pS396-Tau and predicted their abundance in retinal cross sections. For the first time, we reported the spectral signature of pTau and demonstrated an accurate prediction of Aβ and pTau distribution powered by deep learning. We expect our finding will lay the groundwork for label-free detection of AD at its very earliest roots.Significance StatementThe pathological hallmarks of Alzheimer’s disease (AD), amyloid β-protein (Aβ) and hyperphosphorylated (p)Tau protein have been characterized by a hyperspectral camera in terms of spectral signatures. The unique optical properties of the hallmark proteins on the broad visible light range enable label-free and high-resolution detection and virtual staining of abnormal deposition in the retina tissue, which will lay the groundwork for AD early diagnosis and AD development quantification.

The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(β-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aβ/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways.

Pericyte loss and deficient vascular platelet-derived growth factor receptor-β (PDGFRβ) signaling are prominent features of the blood–brain barrier breakdown described in Alzheimer’s disease (AD) that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid β-protein (Aβ) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aβ deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRβ in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ40 and Aβ42 burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRβ and Aβ40 levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRβ loss accompanying increased vascular amyloidosis in Alzheimer’s retina implies compromised blood–retinal barrier integrity and provides new targets for AD diagnosis and therapy. Electronic supplementary material The online version of this article (10.1007/s00401-020-02134-w) contains supplementary material, which is available to authorized users.

Visual impairments, notably loss of contrast sensitivity and color vision, were documented in Alzheimer’s disease (AD) patients yet are critically understudied. This protocol describes a novel visual-stimuli four-arm maze (ViS4M; also called visual x-maze), which is a versatile x-shaped maze equipped with spectrum- and intensity-controlled light-emitting diode (LED) sources and dynamic grayscale objects. The ViS4M is designed to allow the assessment of color and contrast vision along with locomotor and cognitive functions in mice. In the color testing mode, the spectral distributions of the LED lights create four homogenous spaces that differ in chromaticity and luminance, corresponding to the mouse visual system. In the contrast sensitivity test, the four grayscale objects are placed in the middle of each arm, contrasting against the black walls and the white floors of the maze. Upon entering the maze, healthy wild-type (WT) mice tend to spontaneously alternate between arms, even under equiluminant conditions of illumination, suggesting that cognitively and visually intact mice use both color and brightness as cues to navigate the maze. Evaluation of the double-transgenic APP(SWE)/PS1(ΔE9) mouse model of AD (AD(+) mice) reveals substantial deficits to alternate in both color and contrast modes at an early age, when hippocampal-based memory and learning is still intact. Profiling of timespan, entries, and transition patterns between the different arms uncovers variable aging and AD-associated impairments in color discrimination and contrast sensitivity. The analysis of arm sequences of alternation reveals different pathways of exploration in young WT, old WT, and AD(+) mice, which can be used as color and contrast imprints of functionally intact versus impaired mice. Overall, we describe the utility of a novel visual x-maze test to identify behavioral changes in mice related to cognition, as well as color and contrast vision, with high precision and reproducibility. Graphic abstract: [Image: see text] Exploratory behavior of AD(+) mice versus age- and sex-matched WT mice is tracked (top left: trajectory from a 5-min video file) in a novel visual-stimuli four-arm maze (ViS4M; also named visual x-maze) equipped with spectrum- and intensity-controlled LED sources or grayscale objects. Consecutive arm entries reveal that APP(SWE)/PS1(ΔE9) (AD(+)) mice alternate less between arms, as opposed to WT mice. Sequence analysis, according to the three alternation pathways (depicted by white, yellow, and brown arrows) under different conditions of illumination, uncovers specific deficits linked to color vision in AD(+) mice, evidenced by a color imprint chart.

Extensive effort has been made studying retinal pathology in Alzheimer’s disease (AD) to improve early noninvasive diagnosis and treatment. Particularly relevant are vascular changes, which appear prominent in early brain pathogenesis and could predict cognitive decline. Recently, we identified platelet-derived growth factor receptor beta (PDGFRβ) deficiency and pericyte loss associated with vascular Aβ deposition in the neurosensory retina of mild cognitively impaired (MCI) and AD patients. However, the pathological mechanisms of retinal vascular changes and their possible relationships with vascular amyloidosis, pericyte loss, and blood-retinal barrier (BRB) integrity remain unknown. Here, we evaluated the retinas of transgenic APPSWE/PS1ΔE9 mouse models of AD (ADtg mice) and wild-type mice at different ages for capillary degeneration, PDGFRβ expression, vascular amyloidosis, permeability and inner BRB tight-junction molecules. Using a retinal vascular isolation technique followed by periodic acid-Schiff or immunofluorescent staining, we discovered significant retinal capillary degeneration in ADtg mice compared to age- and sex-matched wild-type mice (P P = 0.0035), with males more susceptible than females. Degeneration of retinal capillaries also progressively increased with age in healthy mice (P = 0.0145); however, the phenomenon was significantly worse during AD-like progression (P = 0.0001). A substantial vascular PDGFRβ deficiency (~ 50% reduction, P = 0.0017) along with prominent vascular Aβ deposition was further detected in the retina of ADtg mice, which inversely correlated with the extent of degenerated capillaries (Pearson’s r = − 0.8, P = 0.0016). Importantly, tight-junction alterations such as claudin-1 downregulation and increased BRB permeability, demonstrated in vivo by retinal fluorescein imaging and ex vivo following injection of FITC-dextran (2000 kD) and Texas Red-dextran (3 kD), were found in ADtg mice. Overall, the identification of age- and Alzheimer’s-dependent retinal capillary degeneration and compromised BRB integrity starting at early disease stages in ADtg mice could contribute to the development of novel targets for AD diagnosis and therapy.

We introduce a novel visual-stimuli four-arm maze (ViS4M) equipped with spectrally- and intensity-controlled LED emitters and dynamic grayscale objects that relies on innate exploratory behavior to assess color and contrast vision in mice. Its application to detect visual impairments during normal aging and over the course of Alzheimer’s disease (AD) is evaluated in wild-type (WT) and transgenic APPSWE/PS1∆E9 murine models of AD (AD+) across an array of irradiance, chromaticity, and contrast conditions. Substantial color and contrast-mode alternation deficits appear in AD+ mice at an age when hippocampal-based memory and learning is still intact. Profiling of timespan, entries and transition patterns between the different arms uncovers variable AD-associated impairments in contrast sensitivity and color discrimination, reminiscent of tritanomalous defects documented in AD patients. Transition deficits are found in aged WT mice in the absence of alternation decline. Overall, ViS4M is a versatile, controlled device to measure color and contrast-related vision in aged and diseased mice.

Despite growing evidence for the characteristic signs of Alzheimer's disease (AD) in the neurosensory retina, our understanding of retina–brain relationships, especially at advanced disease stages and in response to therapy, is lacking. In transgenic models of AD (APPSWE/PS1∆E9; ADtg mice), glatiramer acetate (GA) immunomodulation alleviates disease progression in pre‐ and early‐symptomatic disease stages. Here, we explored the link between retinal and cerebral AD‐related biomarkers, including response to GA immunization, in cohorts of old, late‐stage ADtg mice. This aged model is considered more clinically relevant to the age‐dependent disease. Levels of synaptotoxic amyloid β‐protein (Aβ)1–42, angiopathic Aβ1–40, non‐amyloidogenic Aβ1–38, and Aβ42/Aβ40 ratios tightly correlated between paired retinas derived from oculus sinister (OS) and oculus dexter (OD) eyes, and between left and right posterior brain hemispheres. We identified lateralization of Aβ burden, with one‐side dominance within paired retinal and brain tissues. Importantly, OS and OD retinal Aβ levels correlated with their cerebral counterparts, with stronger contralateral correlations and following GA immunization. Moreover, immunomodulation in old ADtg mice brought about reductions in cerebral vascular and parenchymal Aβ deposits, especially of large, dense‐core plaques, and alleviation of microgliosis and astrocytosis. Immunization further enhanced cerebral recruitment of peripheral myeloid cells and synaptic preservation. Mass spectrometry analysis identified new parallels in retino‐cerebral AD‐related pathology and response to GA immunization, including restoration of homeostatic glutamine synthetase expression. Overall, our results illustrate the viability of immunomodulation‐guided CNS repair in old AD model mice, while shedding light onto similar retino‐cerebral responses to intervention, providing incentives to explore retinal AD biomarkers., In this study, Doustar et al. revealed that retinal Abeta burden predicts its brain levels in old, late‐stage murine models of Alzheimer's disease and further in response to immunotherapy. Substantial therapeutic effects are detected even at such advanced disease stage; immunomodulation effectively mitigates vascular and parenchymal amyloid‐beta deposition, diminishes neuroinflammation, as well as restores synaptic density and retino‐cerebral glutamine synthetase levels.

Additional file 3. Supplementary Figures 1–5.

Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-β protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer’s-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-β1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-β1-42, vascular and parenchymal amyloid-β deposits, and astrocytosis (31%, 47–80%, and 33%, respectively; P < 0.05–0.0001). ACE10 macrophages surrounded brain and retinal amyloid-β plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05–0.001). In vitro analysis of macrophage response to well-defined amyloid-β1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-β1-42 species by ACE10 macrophages. They exhibited 2–5-fold increased surface binding to amyloid-β conformers as well as substantially more effective amyloid-β1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05–0.0001), endosomal processing (P < 0.05–0.0001), and ∼80% increased extracellular degradation of amyloid-β1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer’s-related amyloid-β1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-β forms.

Osteopontin (OPN), a matricellular immunomodulatory cytokine highly expressed by myelomonocytic cells, is known to regulate immune cell migration, communication, and response to brain injury. Enhanced cerebral recruitment of monocytes achieved through glatiramer acetate (GA) immunization or peripheral blood enrichment with bone marrow (BM)-derived CD115+ monocytes (MoBM) curbs amyloid β-protein (Aβ) neuropathology and preserves cognitive function in murine models of Alzheimer's disease (ADtg mice). To elucidate the beneficial mechanisms of these immunomodulatory approaches in AD, we focused on the potential role of OPN in macrophage-mediated Aβ clearance. Here, we found extensive OPN upregulation along with reduction of vascular and parenchymal Aβ burden in cortices and hippocampi of GA-immunized ADtg mice. Treatment combining GA with blood-grafted MoBM further increased OPN levels surrounding residual Aβ plaques. In brains from AD patients and ADtg mice, OPN was also elevated and predominantly expressed by infiltrating GFP+- or Iba1+-CD45high monocyte-derived macrophages engulfing Aβ plaques. Following GA immunization, we detected a significant increase in a subpopulation of inflammatory blood monocytes (CD115+CD11b+Ly6Chigh) expressing OPN, and subsequently, an elevated population of OPN-expressing CD11b+Ly6C+CD45high monocyte/macrophages in the brains of these ADtg mice. Correlogram analyses indicate a strong linear correlation between cerebral OPN levels and macrophage infiltration, as well as a tight inverse relation between OPN and Aβ-plaque burden. In vitro studies corroborate in vivo findings by showing that GA directly upregulates OPN expression in BM-derived macrophages (MФBM). Further, OPN promotes a phenotypic shift that is highly phagocytic (increased uptake of Aβ fibrils and surface scavenger receptors) and anti-inflammatory (altered cell morphology, reduced iNOS, and elevated IL-10 and Aβ-degrading enzyme MMP-9). Inhibition of OPN expression in MФBM, either by siRNA, knockout (KOOPN), or minocycline, impairs uptake of Aβ fibrils and hinders GA's neuroprotective effects on macrophage immunological profile. Addition of human recombinant OPN reverses the impaired Aβ phagocytosis in KOOPN-MФBM. This study demonstrates that OPN has an essential role in modulating macrophage immunological profile and their ability to resist pathogenic forms of Aβ.

Background Mechanical ventilation is strongly associated with cognitive decline after critical illness. This finding is particularly evident among older individuals who have pre-existing cognitive impairment, most commonly characterized by varying degrees of cerebral amyloid-β accumulation, neuroinflammation, and blood-brain barrier dysfunction. We sought to test the hypothesis that short-term mechanical ventilation contributes to the neuropathology of cognitive impairment by (i) increasing cerebral amyloid-β accumulation in mice with pre-existing Alzheimer’s disease pathology, (ii) increasing neurologic and systemic inflammation in wild-type mice and mice with pre-existing Alzheimer’s disease pathology, and (iii) increasing hippocampal blood-brain barrier permeability in wild-type mice and mice with pre-existing Alzheimer’s disease pathology. Methods We subjected double transgenic Alzheimer’s disease (APP/PSEN1) and wild-type mice to mechanical ventilation for 4 h and compared to non-mechanically ventilated Alzheimer’s disease model and wild-type mice. Cerebral soluble/insoluble amyloid-β1–40/amyloid-β1–42 and neurological and systemic markers of inflammation were quantified. Hippocampal blood-brain barrier permeability was quantified using a novel methodology that enabled assessment of small and large molecule permeability across the blood-brain barrier. Results Mechanical ventilation resulted in (i) a significant increase in cerebral soluble amyloid-β1–40 (p = 0.007) and (ii) significant increases in neuroinflammatory cytokines in both wild-type and Alzheimer’s disease mice which, in most cases, were not reflected in the plasma. There were (i) direct correlations between polymorphonuclear cells in the bronchoalveolar fluid and cerebral soluble amyloid-β1–40 (p = 0.0033), and several Alzheimer’s disease-relevant neuroinflammatory biomarkers including cerebral TNF-α and IL-6; (iii) significant decreases in blood-brain barrier permeability in mechanically ventilated Alzheimer’s disease mice and a trend towards increased blood-brain barrier permeability in mechanically ventilated wild-type mice. Conclusions These results provide the first evidence that short-term mechanical ventilation independently promotes the neuropathology of Alzheimer’s disease in subjects with and without pre-existing cerebral Alzheimer’s disease pathology. Future studies are needed to further clarify the specific mechanisms by which this occurs and to develop neuroprotective mechanical ventilation strategies that mitigate the risk of cognitive decline after critical illness. Electronic supplementary material The online version of this article (10.1186/s13054-019-2356-2) contains supplementary material, which is available to authorized users.

Figure S1. Mechanical ventilation-associated lung injury and cerebral Aβ concentrations in wild-type (WT) and transgenic Alzheimer’s Disease (ADtg) mice. Figure S2. Key cytokines in plasma of WT and ADtg following mechanical ventilation (MV). The Meso Scale Discovery (MSD) multiplex inflammatory assay performed on plasma and brain. Figure S3. IL-12p70 and IL-2. Figure S4. IFNγ and IL-4 cytokines. Figure S5. IL-15, IL-33, and IL-17A/F. Figure S6. MIP-1α, MCP-1 and MIP-2. Figure S7. KC/GRO and IP-10. Figure S8. Correlations between cerebral soluble Aβ and key brain cytokines in ADtg mice and correlations between soluble Aβ1–40 and Aβ1–42, and cerebral cytokines from age-matched ADtg mice subjected to MV or SB. Figure S9. Correlations between cerebral soluble Aβ and chemokines in SB and MV ADtg mice. Correlation between soluble Aβ1–40 and Aβ1–42 levels and the cerebral inflammatory cytokines performed from age-matched ADtg mice. Figure S10. Correlations between cerebral insoluble Aβ and cerebral inflammatory biomarkers in SB and MV ADtg mice. Correlation between insoluble Aβ1–40 and Aβ1–42 and inflammatory cytokines from brains of ADtg mice subjected to MV or SB. Figure S11. Hippocampal blood-brain barrier permeability of WT and ADtg mice following MV. A. Representative confocal images of Texas Red-dextran tracer in WT (top) and ADtg (bottom) mice under SB condition. B. Correlation between Texas Red-dextran hippocampal leakage and PMNs in the bronchoalveolar lavage fluid. C-D. Correlation between soluble Aβ1–40 in age-matched ADtg mice, and C. FITC-dextran hippocampal leakage, and D. Texas Red-dextran hippocampal leakage. E. Correlation of soluble Aβ1–42 in age-matched ADtg mice and Texas Red-dextran hippocampal permeability. F-G. Correlation between insoluble Aβ1–40 in ADtg mice, and F. FITC-dextran hippocampal leakage, and G. Texas Red-dextran hippocampal leakage. H-I. Correlation between insoluble Aβ1–42 and: H. FITC-dextran hippocampal leakage, and I. Texas Red-dextran hippocampal permeability. (PDF 8663 kb)

Background Noninvasive detection of Alzheimer's disease (AD) with high specificity and sensitivity can greatly facilitate identification of at-risk populations for earlier, more effective intervention. AD patients exhibit a myriad of retinal pathologies, including hallmark amyloid β-protein (Aβ) deposits. Methods Burden, distribution, cellular layer, and structure of retinal Aβ plaques were analyzed in flat mounts and cross sections of definite AD patients and controls (n = 37). In a proof-of-concept retinal imaging trial (n = 16), amyloid probe curcumin formulation was determined and protocol was established for retinal amyloid imaging in live patients. Results Histological examination uncovered classical and neuritic-like Aβ deposits with increased retinal Aβ42 plaques (4.7-fold; P = 0.0063) and neuronal loss (P = 0.0023) in AD patients versus matched controls. Retinal Aβ plaque mirrored brain pathology, especially in the primary visual cortex (P = 0.0097 to P = 0.0018; Pearson's r = 0.84-0.91). Retinal deposits often associated with blood vessels and occurred in hot spot peripheral regions of the superior quadrant and innermost retinal layers. Transmission electron microscopy revealed retinal Aβ assembled into protofibrils and fibrils. Moreover, the ability to image retinal amyloid deposits with solid-lipid curcumin and a modified scanning laser ophthalmoscope was demonstrated in live patients. A fully automated calculation of the retinal amyloid index (RAI), a quantitative measure of increased curcumin fluorescence, was constructed. Analysis of RAI scores showed a 2.1-fold increase in AD patients versus controls (P = 0.0031). Conclusion The geometric distribution and increased burden of retinal amyloid pathology in AD, together with the feasibility to noninvasively detect discrete retinal amyloid deposits in living patients, may lead to a practical approach for large-scale AD diagnosis and monitoring. Funding National Institute on Aging award (AG044897) and The Saban and The Marciano Family Foundations.

Cognitive decline in patients with Alzheimer's disease (AD) is associated with elevated brain levels of amyloid β protein (Aβ), particularly neurotoxic Aβ(1-42). Angiotensin-converting enzyme (ACE) can degrade Aβ(1-42), and ACE overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE(10/10) mice, which overexpress ACE in myelomonocytes using the c-fms promoter, with the transgenic APP(SWE)/PS1(ΔE9) mouse model of AD (AD⁺). Evaluation of brain tissue from these AD⁺ACE(10/10) mice at 7 and 13 months revealed that levels of both soluble and insoluble brain Aβ(1-42) were reduced compared with those in AD⁺ mice. Furthermore, both plaque burden and astrogliosis were drastically reduced. Administration of the ACE inhibitor ramipril increased Aβ levels in AD⁺ACE(10/10) mice compared with the levels induced by the ACE-independent vasodilator hydralazine. Overall, AD⁺ACE(10/10) mice had less brain-infiltrating cells, consistent with reduced AD-associated pathology, though ACE-overexpressing macrophages were abundant around and engulfing Aβ plaques. At 11 and 12 months of age, the AD⁺ACE(10/WT) and AD⁺ACE(10/10) mice were virtually equivalent to non-AD mice in cognitive ability, as assessed by maze-based behavioral tests. Our data demonstrate that an enhanced immune response, coupled with increased myelomonocytic expression of catalytically active ACE, prevents cognitive decline in a murine model of AD.

Weekly glatiramer acetate immunization of transgenic mice modelling Alzheimer's disease resulted in retained cognition (Morris water maze test), decreased amyloid-β plaque burden, and regulation of local inflammation through a mechanism involving enhanced recruitment of monocytes. Ablation of bone marrow-derived myeloid cells exacerbated plaque pathology, whereas weekly administration of glatiramer acetate enhanced cerebral recruitment of innate immune cells, which dampened the pathology. Here, we assessed the therapeutic potential of grafted CD115(+) monocytes, injected once monthly into the peripheral blood of transgenic APPSWE/PS1ΔE9 Alzheimer's disease mouse models, with and without weekly immunization of glatiramer acetate, as compared to glatiramer acetate alone. All immune-modulation treatment groups were compared with age-matched phosphate-buffered saline-injected control transgenic and untreated non-transgenic mouse groups. Two independent cohorts of mice were assessed for behavioural performance (6-8 mice/group); treatments started in 10-month-old symptomatic mice and spanned a total of 2 months. For all three treatments, our data suggest a substantial decrease in cognitive deficit as assessed by the Barnes maze test (P < 0.0001-0.001). Improved cognitive function was associated with synaptic preservation and reduction in cerebral amyloid-β protein levels and astrogliosis (P < 0.001 and P < 0.0001), with no apparent additive effects for the combined treatment. The peripherally grafted, green fluorescent protein-labelled and endogenous monocytes, homed to cerebral amyloid plaques and directly engulfed amyloid-β; their recruitment was further enhanced by glatiramer acetate. In glatiramer acetate-immunized mice and, moreover, in the combined treatment group, monocyte recruitment to the brain was coupled with greater elevation of the regulatory cytokine IL10 surrounding amyloid-β plaques. All treated transgenic mice had increased cerebral levels of MMP9 protein (P < 0.05), an enzyme capable of degrading amyloid-β, which was highly expressed by the infiltrating monocytes. In vitro studies using primary cultures of bone marrow monocyte-derived macrophages, demonstrated that glatiramer acetate enhanced the ability of macrophages to phagocytose preformed fibrillar amyloid-β1-42 (P < 0.0001). These glatiramer acetate-treated macrophages exhibited increased expression of the scavenger receptors CD36 and SCARA1 (encoded by MSR1), which can facilitate amyloid-β phagocytosis, and the amyloid-β-degrading enzyme MMP9 (P < 0.0001-0.001). Overall, our studies indicate that increased cerebral infiltration of monocytes, either by enrichment of their levels in the circulation or by weekly immunization with glatiramer acetate, resulted in substantial attenuation of disease progression in murine Alzheimer's models by mechanisms that involved enhanced cellular uptake and enzymatic degradation of toxic amyloid-β as well as regulation of brain inflammation.

The cognitive decline in Alzheimer's disease (AD) is associated with elevated brain levels of amyloid β-protein (Aβ), particularly Aβ 1-42 . Angiotensin-converting enzyme (ACE) can enzymatically degrade Aβ 1-42 and its overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE 10/10 mice, which over express ACE in myelomonocytic cells, with the double-transgenic APP SWE /PS1 ΔE9 mouse model of AD (AD + ). At 7 months, soluble Aβ 1-42 was reduced by 44% (136 vs 245 pg/mg) and Aβ 1-40 was reduced by 32% (21 vs 31 pg/mg). Plaque burden was reduced by as much as 79% (13 vs 62 10 3 mm 2 ) and insoluble Aβ 1-42 by 64% (189 vs 521 pg/mg). There was also a substantial reduction in astrogliosis (49-57% at 7 months, 50% at 13 months). AD + ACE 10/10 mice demonstrated less overall brain infiltrating cells, consistent with less AD pathology, though ACE-overexpressing monocytes and macrophages were increasingly abundant surrounding and engulfing Aβ plaque. At 11 and 12 months, AD + ACE 10/WT and AD + ACE 10/10 mice were virtually equivalent to non-AD mice in cognitive ability as assessed by maze-based behavioral tests. This study shows that an enhanced immune response, coupled with increased myelomonocytic cell expression of ACE, resulted in essentially complete prevention of the cognitive decline observed in a murine model of AD.

Background: Previous studies from our group and others have demonstrated that peripheral immune cells and especially infiltrating bloodborne monocytes can home to pathological beta-amyloid protein aggregates (Ab plaques) and facilitate their clearance by phagocytosis and secretion of Ab-degrading enzymes. One of the enzymes that was shown capable of degrading Ab and convert the deleterious Ab (1-42) to shorter nontoxic isoforms is angiotensin-converting enzyme (ACE). Results: We found a marked reduction in Ab plaque number and size in the cortex and hippocampus of 5, 7 and 12 month-old AD mice; above 40% decrease in mice having one ACE10 allele and above 80% in mice having two ACE10 alleles. Moreover, the levels of soluble neurotoxic Ab (1-42) isoforms were substantially decreased in brain and plasma of ACE10xADtg mice. Importantly, ADtg mice carrying either one or two ACE10 alleles had a considerable improvement in learning and memory functions as assessed by a panel of behavioral tests such as the Barnes maze, and were indistinguishable than the wild type non-AD mice. These effects were accompanied by an extensive reduction in cortical astrogliosis as assessed by number and area of reactive GFAP-positive astrocytes. Flow cytometry analysis of immune cell profiles in the brain revealed a significant increase in migration of ACE10-overexpessing monocytes as compared to wt monocytes in ADtg mice. Conclusions: These studies indicate that selective ACE-overexpression in microglia and monocytes leads to a remarkable attenuation of AD-like progression in mouse models. Therefore, targeted overexpression of ACE on monocytes may present a highly promising and novel disease-modifying approach for AD treatment.