Dieu-Trang Fuchs

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.

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.

A novel role for osteopontin in macrophage-mediated amyloid-β clearance in Alzheimer’s models

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 Alzheimers 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 GAs 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.

IMMUNOMODULATORY FACTOR INDUCED BY GA MEDIATES MACROPHAGE CLEARANCE OF PATHOGENIC AB FORMS AND ENHANCES SYNAPTIC PRESERVATION

lesions and showed no AD-related lesions or only sparse induction of amyloidosis. However, none evaluated the impact of AD-brain inoculation on other AD hallmarks such as cognitive decline. Our main objective was therefore to determine the impacts of experimental transmission on both brain function and integrity in mouse lemur primates (Microcebus murinus). Methods:Mouse lemurs are mouse-like sized primates that can sometimes spontaneously develop cognitive impairments, amyloidosis, tauopathy and/or cerebral atrophy while aging (>6 years old). Twelve adult mouse lemurs, aged from 3 to 4 years old, were included in this study. Human brain homogenates from AD patients or control (CTRL) age-matched subject were bilaterally inoculated in the hippocampus and subjacent cortex (n1⁄46/group; 4x6.25ml; 10% weight/volume). We performed longitudinal cognitive, electroencephalography (EEG) and morphological MRI studies up to 18 months post-inoculation followed by immunohistopathological examination of brain tissues. Results:AD-inoculated animals progressively developed cognitive impairments affecting first long-term memory and later learning abilities. EEG profiles of AD-inoculated animals progressively shifted towards fast frequencies compared to CTRLinoculated ones indicating neuronal activity alterations. Compared to the CTRL-inoculated group, AD-inoculated animals displayed an atrophy of the retrosplenial and posterior cingulate cortices as well as adjacent regions belonging to the limbic connectome (such as the hippocampus, entorhinal cortex or amygdala). Using immunohistochemistry, no marked AD neuropathological lesions were detected apart from few cortical amyloid plaques and sparse cortical amyloid angiopathy in some AD-inoculated animals (3/6). Conclusions:We have demonstrated for the first time that AD brain homogenates inoculation induces functional and morphological alterations in primates without induction of immunohistopathological-detectable forms of Abeta or tau proteins. This suggests that the most toxic entities induced by AD brain inoculation escape from detection by classical immunohistology technics. This work supported by the France-Alzheimer Association and by the Foundation Plan Alzheimer.

IDENTIFICATION OF INFLAMMATORY PROCESSES SURROUNDING AMYLOID-β DEPOSITS IN THE RETINA OF MCI AND AD PATIENTS

P4-254 IDENTIFICATION OF INFLAMMATORY PROCESSES SURROUNDING AMYLOID-b DEPOSITS INTHERETINAOFMCIANDAD PATIENTS Koronyo-Hamaoui Maya, Altan Rentsendorj, Ernesto Barron, DieuTrang Fuchs, Carol A. Miller, Keith L. Black, David R. Hinton, Giovanni Meli, Yosef Koronyo, Department of Neurosurgery, Maxine Dunitz Neurosurgical Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Biomedical Sciences, Maxine Dunitz Neurosurgical Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Doheney Eye Institute, Los Angeles, CA, USA; Department of Pathology Program in Neuroscience, Keck School of Medicine, USC, Los Angeles, CA, USA; Departments of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, USC, Los Angeles, CA, USA; European Brain Research Institute (EBRI), Rome, Italy. Contact e-mail: Maya.Koronyo@cshs.org

OSTEOPONTIN-DEPENDENT PHAGOCYTOSIS OF SYNAPTOTOXIC Aβ BY INFILTRATING MONOCYTES AND MACROPHAGES

as a master regulator of brain bioenergetics via mitochondrial function. However, the underlying mechanism is not fully elucidated. Interestingly, in neuronal cells, estrogen receptor beta (ERb), but not estrogen receptor alpha (ERa), was demonstrated to co-localize with mitochondria, and has a functional role in ATP production, suggesting potential differential contributions of ERa and ERb to regulation of mitochondrial bioenergetics. Methods: To identify cell type specific responses to estrogen treatment, we used rat embryonic primary neurons and astrocytes. To identify the contribution of ER subtypes, cells were treated with either ERa selective antagonist (MPP), or ERb selective antagonist (PHTPP) prior to E2 treatment. Cellular respiratory capacity was determined by XF24 metabolic flux analyzer, and Oxygen consumption rate (OCR) was used as an indicator for mitochondrial oxidative phosphorylation. To determine whether ERs regulate mitochondrial respiration via regulation of transcription of mitochondrial genome, gene expression of the 13 mitochondrial protein encoding genes will be determined by real-time rtPCR. Ongoing analysis also explores how mitochondrial genetic variances affect treatment effect of selective estrogen modulators on perimenopausal symptoms. Results: Estrogen increased both ATP production and maximum respiration in neurons, but only ATP production in astrocytes. ERa antagonist prevented E2 mediated enhancement of mitochondrial respiration in astrocytes but not neurons, whereas ERb antagonist abolished E2 mediated enhancement of mitochondrial respiration in both cell types. Conclusions:E2 regulation of mitochondrial respiration is cell type and ER subtype specific, possibly through differential regulation of transcription of the mitochondrial genome. Acknowledgement: This work was supported by NIA R01 AG032236 to RDB; NIA P01AG026572 to RDB, Project 1 to RDB & EC.

GENETICALLY MODIFIED ACE10-MACROPHAGES EXHIBIT SUPERIOR CAPACITY TO ERADICATE STRUCTURALLY DEFINED AB FORMS LINKED TO ALZHEIMER’S DISEASE

Background: TgAD mouse models of Alzheimer’s disease treated with scyllo-inositol showed reduced amyloid load and reversed behavioural changes (McLaurin et al 2006 Nat Med). scyllo-Inositol is a competitive inhibitor of myo-inositol for transport across the blood brain barrier leading to reduction in intracellular and brain myo-inositol levels (Fenili et al 2011 PLOSONE). Clinical trial results indicate scyllo-inositol treatment reduced agitation and aggression in patients with Alzheimer’s disease. The mechanism of action has yet to be proven, although reduction of brain myoinositol concentration is a candidate. Methods: Gene expression for TgAD and nonTg littermate mice treated with scyllo-inositol were compared to untreated control mice (n1⁄43) using Affymetrix mouse genome 430 microarrays. The data (FDR1⁄40.05 FC>2) was analyzed using The Database for Annotation, Visualization and Integrated Discovery (DAVID) and Qiagen’s Ingenuity Pathway Analysis. Results:Three comparisons were analyzed, nontransgenic (nTg) hippocampus, nTg cortex, and transgenic (Tg) hippocampus. nTg hippocampal gene expression identified 596 probe sets that met the predetermined cut-offs, nTg cortical gene expression identified 725, and Tg hippocampal gene expression identified 541 probe sets. nTg cortex differentially expressed only 18 probe sets between treated and untreated mice, thus analysis was not performed. Synaptic transmission was a common enriched function from DAVID for all three comparisons. From Ingenuity Pathway Analysis, the common top canonical pathway affected by scyllo-inositol treatment in hippocampus and cortex of nTg is glutamate receptor signalling, which is not affected in Tg hippocampus. Common top upstream regulators between all three comparisons were BDNF and HTT. Conclusions: scyllo-Inositol treatment modulates gene expression differentially in TgAD versus nTgmice suggesting that treatment has Ab-dependent and independent effects. Ingenuity Pathways Analysis identified gene expression changes in TgAD mice that have been previously linked to Alzheimer’s disease patients, thus supporting the pathways identified in this analysis.

Rescue of synapses with glatiramer acetate immunotherapy in a murine model of Alzheimer's disease

O2-05-05 RESCUE OF SYNAPSES WITH GLATIRAMER ACETATE IMMUNOTHERAPY IN A MURINE MODEL OFALZHEIMER’S DISEASE Songlin Li, David A. Daley, Yosef Koronyo, Dieu-Trang Fuchs, Julia Sheyn, Altan Rentsendorj, Eric Y. Hayden, Keith L. Black, David B. Teplow, Koronyo-Hamaoui Maya, Cedars-Sinai Medical Center, Los Angeles, CA, USA; David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Contact e-mail: Songlin.Li@cshs.org

Targeted high-expression of ACE to myelomonocytic cells diminishes Alzheimer's-like pathology and restores memory and learning in mouse models

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.