Brenda C. Salumbides

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.

Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism

BackgroundSuccinate dehydrogenase (SDH) is a mitochondrial metabolic enzyme complex involved in both the electron transport chain and the citric acid cycle. SDH mutations resulting in enzymatic dysfunction have been found to be a predisposing factor in various hereditary cancers. Therefore, SDH has been implicated as a tumor suppressor.ResultsWe identified that dysregulation of SDH components also occurs in serous ovarian cancer, particularly the SDH subunit SDHB. Targeted knockdown of Sdhb in mouse ovarian cancer cells resulted in enhanced proliferation and an epithelial-to-mesenchymal transition (EMT). Bioinformatics analysis revealed that decreased SDHB expression leads to a transcriptional upregulation of genes involved in metabolic networks affecting histone methylation. We confirmed that Sdhb knockdown leads to a hypermethylated epigenome that is sufficient to promote EMT. Metabolically, the loss of Sdhb resulted in reprogrammed carbon source utilization and mitochondrial dysfunction. This altered metabolic state of Sdhb knockdown cells rendered them hypersensitive to energy stress.ConclusionsThese data illustrate how SDH dysfunction alters the epigenetic and metabolic landscape in ovarian cancer. By analyzing the involvement of this enzyme in transcriptional and metabolic networks, we find a metabolic Achilles’ heel that can be exploited therapeutically. Analyses of this type provide an understanding how specific perturbations in cancer metabolism may lead to novel anticancer strategies.

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.

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.

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.

The TNF family member TL1A induces IL-22 secretion in committed human T h 17 cells via IL-9 induction.

TL1A contributes to the pathogenesis of several chronic inflammatory diseases, including those of the bowel by enhancing TH1, TH17, and TH2 responses. TL1A mediates a strong costimulation of these TH subsets, particularly of mucosal CCR9+ T cells. However, the signaling pathways that TL1A induces in different TH subsets are incompletely understood. We investigated the function of TL1A on human TH17 cells. TL1A, together with TGF‐β, IL‐6, and IL‐23, enhanced the secretion of IL‐17 and IFN‐γ from human CD4+ memory T cells. TL1A induced expression of the transcription factors BATF and T‐bet that correlated with the secretion of IL‐17 and IFN‐γ. In contrast, TL1A alone induced high levels of IL‐22 in memory CD4+ T cells and committed TH17 cells. However, TL1A did not enhance expression of IL‐17A in TH17 cells. Expression of the transcription factor aryl hydrocarbon receptor, which regulates the expression of IL‐22 was not affected by TL1A. Transcriptome analysis of TH17 cells revealed increased expression of IL‐9 in response to TL1A. Blocking IL‐9 receptor antibodies abrogated TL1A‐induced IL‐22 secretion. Furthermore, TL1A increased IL‐9 production by peripheral TH17 cells isolated from patients with Crohn’s disease. These data suggest that TL1A differentially induces expression of TH17 effector cytokines IL‐17, ‐9, and ‐22 and provides a potential target for therapeutic intervention in TH17‐driven chronic inflammatory diseases.

The retina as a window to monitor Alzheimer's disease in human and rodent models: In vivo imaging of retinal Aβ plaques for immunotherapy assessment

a significantly lower Glu/Cr ratio was observed in AD (0.49 6 0.04) compared to CN (0.596 0.03, p<0.05). Meanwhile, the NAA/Cr ratio was significantly lower (ps<0.001) in AD than in CN in both the PCG (D1⁄4 0.126 0.02) and the DLPFC (D 1⁄4 0.18 6 0.03). Using solely the absolute metabolite levels of Cr, NAA, and Glu in the DLPFC, the accuracy for the identification of individuals with early AD (vs. CN) reached 0.84 (95% CI 1⁄4 0.74-0.93). Conclusions: An increased Cr concentration in PCG may be associated with the development of AD, reflecting the underlying neurodegenerative pathology and the disturbed balance of energy supply and energy demand. The study also suggests that combining information on changes of Cr levels and those of other major metabolites can help improve AD identification.

Abstract 1088: HOXB13 inhibition of succinate dehydrogenase leads to epithelial-to-mesenchymal transition in mouse ovarian cancer cell lines.

HOXB13 is a homeobox gene whose dysregulation has been implicated in cancer. We have previously shown that HOXB13 plays an oncogenic role in ovarian cancer by driving epithelial-to-mesenchymal transition (EMT); however the mechanism by which this occurs has not been established. Global metabolomics profiling revealed that overexpression of HOXB13 in genetically defined mouse ovarian cancer cell lines inhibits succinate dehydrogenase (SDH) activity. Targeted-knockdown of the SDH subunit SdhB by shRNA results in mislocalization of E-cadherin and a spindle-shaped morphology suggestive of EMT. Metabolomics and real-time flux analysis showed that knockdown of SdhB results in elevated levels of succinate as well as defective mitochondrial respiration. Despite the mitochondrial respiration defect, SdhB knockdown cells display increased anchorage-independent growth in soft agar and enhanced xenograft tumor formation. Elevated levels of succinate have been shown to inhibit α-ketoglutarate requiring enzymes such as the Jumanji C (JmjC)-domain containing H3K27 demethylases, UTX and JMJD3. Indeed, knockdown of SdhB results in a hypermethylated epigenome as evidenced by increased levels of H3K27me2 and H3K27me3. Both the epigenetic and EMT phenotypes can be recapitulated by pharmacological inhibition of UTX and JMJD3 in control cells. These data point to a mechanism wherein modulation of oncometabolite levels can affect the epigenome, thereby influencing cancer cell morphology and tumorigenic properties. Citation Format: Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel Gozo, Brenda Salumbides, Laurent Vergnes, John Asara, Karen Reue, Elizabeth Kensicki, Beth Karlan, Sandra Orsulic. HOXB13 inhibition of succinate dehydrogenase leads to epithelial-to-mesenchymal transition in mouse ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1088. doi:10.1158/1538-7445.AM2013-1088

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.