Yosef Koronyo

Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model.

Noninvasive monitoring of β-amyloid (Aβ) plaques, the neuropathological hallmarks of Alzheimers disease (AD), is critical for AD diagnosis and prognosis. Current visualization of Aβ plaques in brains of live patients and animal models is limited in specificity and resolution. The retina as an extension of the brain presents an appealing target for a live, noninvasive optical imaging of AD if disease pathology is manifested there. We identified retinal Aβ plaques in postmortem eyes from AD patients (n=8) and in suspected early stage cases (n=5), consistent with brain pathology and clinical reports; plaques were undetectable in age-matched non-AD individuals (n=5). In APP(SWE)/PS1(∆E9) transgenic mice (AD-Tg; n=18) but not in non-Tg wt mice (n=10), retinal Aβ plaques were detected following systemic administration of curcumin, a safe plaque-labeling fluorochrome. Moreover, retinal plaques were detectable earlier than in the brain and accumulated with disease progression. An immune-based therapy effective in reducing brain plaques, significantly reduced retinal Aβ plaque burden in immunized versus non-immunized AD mice (n=4 mice per group). In live AD-Tg mice (n=24), systemic administration of curcumin allowed noninvasive optical imaging of retinal Aβ plaques in vivo with high resolution and specificity; plaques were undetectable in non-Tg wt mice (n=11). Our discovery of Aβ specific plaques in retinas from AD patients, and the ability to noninvasively detect individual retinal plaques in live AD mice establish the basis for developing high-resolution optical imaging for early AD diagnosis, prognosis assessment and response to therapies.

Alzheimer's disease in the retina: imaging retinal aβ plaques for early diagnosis and therapy assessment.

Background: Definite Alzheimer’s disease (AD) diagnosis at early stages is vital for targeting intervention, yet currently unavailable. Noninvasive detection of the pathological hallmark, amyloid-β protein (Aβ) plaques, is limited in the brain. However, the existence of Aβ plaques in the retina, possibly at presymptomatic stages, may improve early detection of AD. Objective: To summarize clinical and preclinical evidence showing that the retina, an accessible part of the central nervous system, displays abnormalities in AD, especially Aβ plaque pathology. The ability to monitor in vivo retinal plaque dynamics in response to immunotherapy is also assessed. Methods: Literature analysis of retinal AD pathology and imaging is provided. In our studies, systemic curcumin is administered to enable monitoring of retinal Aβ plaques in live APPSWE/PS1ΔE9 transgenic mice by optical imaging. Results: Visual and retinal abnormalities, including early manifestation of retinal Aβ plaque pathology, have been documented in AD patients and animal models. In mouse models, retinal Aβ plaques accumulate with age and decrease in response to immunotherapy, consistent with brain pathology. Here, we demonstrate that retinal plaques can be individually monitored in real time following glatiramer acetate immunization. Conclusion: Translation of noninvasive retinal-plaque imaging to humans could eventually facilitate early and accurate AD diagnosis and therapy assessment.

Melanopsin retinal ganglion cell loss in Alzheimer disease

Melanopsin retinal ganglion cells (mRGCs) are photoreceptors driving circadian photoentrainment, and circadian dysfunction characterizes Alzheimer disease (AD). We investigated mRGCs in AD, hypothesizing that they contribute to circadian dysfunction.

Attenuation of AD―like neuropathology by harnessing peripheral immune cells: local elevation of IL―10 and MMP―9

Immunization with an altered myelin‐derived peptide (MOG45D) improves recovery from acute CNS insults, partially via recruitment of monocyte‐derived macrophages that locally display a regulatory activity. Here, we investigated the local alterations in the cellular and molecular immunological milieu associated with attenuation of Alzheimer’s disease‐like pathology following immunotherapy. We found that immunization of amyloid precursor protein/presenilin 1 double‐transgenic mice with MOG45D peptide, loaded on dendritic cells, led to a substantial reduction of parenchymal and perivascular amyloid beta (Aβ)‐plaque burden and soluble Aβ(1–42) peptide levels as well as reduced astrogliosis and levels of a key glial scar protein (chondroitin sulphate proteoglycan). These changes were associated with a shift in the local innate immune response, manifested by increased Iba1+/CD45high macrophages that engulfed Aβ, reduced pro‐inflammatory (tumor necrosis factor‐α) and increased anti‐inflammatory (interleukin‐10) cytokines, as well as a significant increase in growth factors (IGF‐1 and TGFβ) in the brain. Furthermore, the levels of matrix metalloproteinase‐9, an enzyme shown to degrade Aβ and is associated with glial scar formation, were significantly elevated in the brain following immunization. Altogether, these results indicate that boosting systemic immune cells leads to a local immunomodulation manifested by elevated levels of anti‐inflammatory cytokines and metalloproteinases that contribute to ameliorating Alzheimer’s disease pathology.

Therapeutic effects of glatiramer acetate and grafted CD115⁺ monocytes in a mouse model of Alzheimer's disease.

Weekly glatiramer acetate immunization of transgenic mice modelling Alzheimers 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 Alzheimers 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 Alzheimers models by mechanisms that involved enhanced cellular uptake and enzymatic degradation of toxic amyloid-β as well as regulation of brain inflammation.

Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer’s disease

BACKGROUND Noninvasive detection of Alzheimers 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; Pearsons 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.

Ocular indicators of Alzheimer’s: exploring disease in the retina

Although historically perceived as a disorder confined to the brain, our understanding of Alzheimer’s disease (AD) has expanded to include extra-cerebral manifestation, with mounting evidence of abnormalities in the eye. Among ocular tissues, the retina, a developmental outgrowth of the brain, is marked by an array of pathologies in patients suffering from AD, including nerve fiber layer thinning, degeneration of retinal ganglion cells, and changes to vascular parameters. While the hallmark pathological signs of AD, amyloid β-protein (Aβ) plaques and neurofibrillary tangles (NFT) comprising hyperphosphorylated tau (pTau) protein, have long been described in the brain, identification of these characteristic biomarkers in the retina has only recently been reported. In particular, Aβ deposits were discovered in post-mortem retinas of advanced and early stage cases of AD, in stark contrast to non-AD controls. Subsequent studies have reported elevated Aβ42/40 peptides, morphologically diverse Aβ plaques, and pTau in the retina. In line with the above findings, animal model studies have reported retinal Aβ deposits and tauopathy, often correlated with local inflammation, retinal ganglion cell degeneration, and functional deficits. This review highlights the converging evidence that AD manifests in the eye, especially in the retina, which can be imaged directly and non-invasively. Visual dysfunction in AD patients, traditionally attributed to well-documented cerebral pathology, can now be reexamined as a direct outcome of retinal abnormalities. As we continue to study the disease in the brain, the emerging field of ocular AD warrants further investigation of how the retina may faithfully reflect the neurological disease. Indeed, detection of retinal AD pathology, particularly the early presenting amyloid biomarkers, using advanced high-resolution imaging techniques may allow large-scale screening and monitoring of at-risk populations.

Angiotensin-converting enzyme overexpression in myelomonocytes prevents Alzheimer’s-like cognitive decline

Cognitive decline in patients with Alzheimers 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.

Egr1 expression is induced following glatiramer acetate immunotherapy in rodent models of glaucoma and Alzheimer's disease.

PURPOSE Immunization with glatiramer acetate (GA) alleviates the neuropathology associated with glaucoma and Alzheimers disease (AD) in rodent models. This research was undertaken to screen for molecular factors underlying GA-induced neuroprotective mechanisms in these models of chronic neurodegeneration. METHODS Gene expression profiles were analyzed in GA-immunized versus nonimmunized elevated-intraocular pressure (IOP) rat models of glaucoma by using whole genome cDNA microarrays and were further validated by quantitative real-time PCR analysis. A gene, prominently upregulated by GA in elevated IOP retina, was further studied in APP(SWE)/PS1(ΔE9)-transgenic (AD-Tg) mice after GA immunization. RESULTS Seven days after treatment with GA, numerous genes were regulated in the retinas of rats with elevated IOP. Comprehensive functional classification and DAVID/KEGG enrichment analysis of GA-induced differentially expressed genes revealed annotation terms and pathways involved in neuroprotection, immune responses, cell communication, and regeneration. Specifically, increased mRNA levels of an early growth response (Egr) 1 gene were evident in GA-immunized retinas with elevated IOP. In AD-Tg mice, a significant increase in hippocampal EGR1 protein levels was also found in response to GA immunization. Nuclear EGR1 in the dentate gyrus colocalized more frequently with doublecortin-positive and Ki67 proliferating neural progenitors in GA-immunized as compared to nonimmunized AD-Tg mice. Further, EGR1 levels were negatively correlated with hippocampal amyloid-β plaque burden. CONCLUSIONS This study presents global gene expression profiles associated with GA immunization in a glaucoma rat model. Moreover, it identifies EGR1 transcription factor as a potential mediator for GA-induced neuroprotection in both glaucoma and AD.

Multimodal wide-field two-photon excitation imaging: Characterization of the technique for in vivo applications

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications.