Georgina V. Bixler

Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

BackgroundDespite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.ResultsUsing the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.ConclusionThese changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment.

BackgroundAge-related cognitive dysfunction, including impairment of hippocampus-dependent spatial learning and memory, affects approximately half of the aged population. Induction of a variety of neuroinflammatory measures has been reported with brain aging but the relationship between neuroinflammation and cognitive decline with non-neurodegenerative, normative aging remains largely unexplored. This study sought to comprehensively investigate expression of the MHC II immune response pathway and glial activation in the hippocampus in the context of both aging and age-related cognitive decline.MethodsThree independent cohorts of adult (12-13 months) and aged (26-28 months) F344xBN rats were behaviorally characterized by Morris water maze testing. Expression of MHC II pathway-associated genes identified by transcriptomic analysis as upregulated with advanced aging was quantified by qPCR in synaptosomal fractions derived from whole hippocampus and in hippocampal subregion dissections (CA1, CA3, and DG). Activation of astrocytes and microglia was assessed by GFAP and Iba1 protein expression, and by immunohistochemical visualization of GFAP and both CD74 (Ox6) and Iba1.ResultsWe report a marked age-related induction of neuroinflammatory signaling transcripts (i.e., MHC II components, toll-like receptors, complement, and downstream signaling factors) throughout the hippocampus in all aged rats regardless of cognitive status. Astrocyte and microglial activation was evident in CA1, CA3 and DG of intact and impaired aged rat groups, in the absence of differences in total numbers of GFAP+ astrocytes or Iba1+ microglia. Both mild and moderate microglial activation was significantly increased in all three hippocampal subregions in aged cognitively intact and cognitively impaired rats compared to adults. Neither induction of MHCII pathway gene expression nor glial activation correlated to cognitive performance.ConclusionsThese data demonstrate a novel, coordinated age-related induction of the MHC II immune response pathway and glial activation in the hippocampus, indicating an allostatic shift toward a para-inflammatory phenotype with advancing age. Our findings demonstrate that age-related induction of these aspects of hippocampal neuroinflammation, while a potential contributing factor, is not sufficient by itself to elicit impairment of spatial learning and memory in models of normative aging. Future efforts are needed to understand how neuroinflammation may act synergistically with cognitive-decline specific alterations to cause cognitive impairment.

Circulating IGF1 regulates hippocampal IGF1 levels and brain gene expression during adolescence

GH and its anabolic mediator, IGF1, are important not only in somatic growth but also in the regulation of brain function. Even though GH treatment has been used clinically to improve body composition and exercise capacity in adults, its influence on central nervous system function has only recently been recognized. This is also the case for children with childhood-onset GH deficiency (GHD) where GH has been used to stimulate bone growth and enhance final adult height. Circulating IGF1 is transported across the blood-brain barrier and IGF1 and its receptors are also synthesized in the brain by neurons and glial and endothelial cells. Nevertheless, the relationship between circulating IGF1 and brain IGF1 remains unclear. This study, using a GH-deficient dwarf rat model and peripheral GH replacement, investigated the effects of circulating IGF1 during adolescence on IGF1 levels in the brain. Our results demonstrated that hippocampal IGF1 protein concentrations during adolescence are highly regulated by circulating IGF1, which were reduced by GHD and restored by systematic GH replacement. Importantly, IGF1 levels in the cerebrospinal fluid were decreased by GHD but not restored by GH replacement. Furthermore, analysis of gene expression using microarrays and RT-PCR indicated that circulating IGF1 levels did not modify the transcription of Igf1 or its receptor in the hippocampus but did regulate genes that are involved in microvascular structure and function, brain development, and synaptic plasticity, which potentially support brain structures involved in cognitive function during this important developmental period.

Hippocampal expression of myelin-associated inhibitors is induced with age-related cognitive decline and correlates with deficits of spatial learning and memory

J. Neurochem. (2012) 121, 77–98.

Multi-Modal Proteomic Analysis of Retinal Protein Expression Alterations in a Rat Model of Diabetic Retinopathy

Background As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. Methodology/Principal Findings A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. Conclusions/Significance These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

A multistep validation process of biomarkers for preclinical drug development.

Biomarkers that can be measured in preclinical models in a high-throughput, reproducible manner offer the potential to increase the speed and efficacy of drug development. Development of therapeutic agents for many conditions is hampered by the limited number of validated preclinical biomarkers available to gauge pharmacoefficacy and disease progression, but the validation process for preclinical biomarkers has received limited attention. This report defines a five-step preclinical biomarker validation process and applies the process to a case study of diabetic retinopathy. By showing that a gene expression panel is highly reproducible, coincides with disease manifestation, accurately classifies individual animals and identifies animals treated with a known therapeutic agent, a biomarker panel can be considered validated. This particular biomarker panel consisting of 14 genes (C1inh, C1s, Carhsp1, Chi3l1, Gat3, Gbp2, Hspb1, Icam1, Jak3, Kcne2, Lama5, Lgals3, Nppa, Timp1) can be used in diabetic retinopathy pharmacotherapeutic research, and the biomarker development process outlined here is applicable to drug development efforts for other diseases.

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome

BackgroundDiabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.MethodsThe retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.ResultsTranscriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.ConclusionsThese results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

Hippocampal Subregions Exhibit Both Distinct and Shared Transcriptomic Responses to Aging and Nonneurodegenerative Cognitive Decline

Impairment of hippocampal-dependent spatial learning and memory with aging affects a large segment of the aged population. Hippocampal subregions (CA1, CA3, and DG) have been previously reported to express both common and specific morphological, functional, and gene/protein alterations with aging and cognitive decline. To comprehensively assess gene expression with aging and cognitive decline, transcriptomic analysis of CA1, CA3, and DG was conducted using Adult (12M) and Aged (26M) F344xBN rats behaviorally characterized by Morris water maze performance. Each subregion demonstrated a specific pattern of responses with aging and with cognitive performance. The CA1 and CA3 demonstrating the greatest degree of shared gene expression changes. Analysis of the pathways, processes, and regulators of these transcriptomic changes also exhibit a similar pattern of commonalities and differences across subregions. Gene expression changes between Aged cognitively Intact and Aged cognitively Impaired rats often showed an inversion of the changes between Adult and Aged rats. This failure to adapt rather than an exacerbation of the aging phenotype questions a conventional view that cognitive decline is exaggerated aging. These results are a resource for investigators studying cognitive decline and also demonstrate the need to individually examine hippocampal subregions in molecular analyses of aging and cognitive decline.

CNS-wide Sexually Dimorphic Induction of the Major Histocompatibility Complex 1 Pathway With Aging

The major histocompatibility complex I (MHCI) pathway, which canonically functions in innate immune viral antigen presentation and detection, is functionally pleiotropic in the central nervous system (CNS). Alternative roles include developmental synapse pruning, regulation of synaptic plasticity, and inhibition of neuronal insulin signaling; all processes altered during brain aging. Upregulation of MHCI components with aging has been reported; however, no systematic examination of MHCI cellular localization, expression, and regulation across CNS regions, life span, and sexes has been reported. In the mouse, MHCI is expressed by neurons and microglia, and MHCI components and receptors (H2-K1, H2-D1, &bgr;2M, Lilrb3, Klra2, CD247) display markedly different expression profiles across the hippocampus, cortex, cerebellum, brainstem, and retina. MHCI components, receptors, associated inflammatory transcripts (IL1&agr;, IL1&bgr;, IL6, TNF&agr;), and TAP (transporter associated with antigen processing) components are induced with aging and to a greater degree in female than male mice across CNS regions. H2-K1 and H2-D1 expression is associated with differential CG and non-CG promoter methylation across CNS regions, ages, and between sexes, and concomitant increased expression of proinflammatory genes. Meta-analysis of human brain aging data also demonstrates age-related increases in MHCI. Induction of MHCI signaling could contribute to altered synapse regulation and impaired synaptic plasticity with aging.

Expression of NgR1-Antagonizing Proteins Decreases with Aging and Cognitive Decline in Rat Hippocampus

The myelin-associated inhibitor/Nogo-66 receptor 1 (NgR1) pathway directly functions in negative modulation of structural and electrophysiological synaptic plasticity. A previous study has established an important role of NgR1 pathway signaling in cognitive function, and we have demonstrated that multiple components of this pathway, including ligands, NgR1 co-receptors, and RhoA, are upregulated at the protein level specifically in cognitively impaired, but not age-matched cognitively intact aged rats. Recent studies have identified two novel endogenous NgR1 antagonists, LOTUS and LGI1, and an alternative co-receptor, ADAM22, which act to suppress NgR1 pathway signaling. To determine whether these endogenous NgR1-inhibiting proteins may play a compensatory role in age-related cognitive impairment by counteracting overexpression of NgR1 agonists and co-receptors, we quantified the expression of LOTUS, LGI1, and ADAM22 in hippocampal CA1, CA3 and DG subregions dissected from mature adult and aged rats cognitively phenotyped for spatial learning and memory by Morris water maze testing. We have found that endogenous inhibitors of NgR1 pathway action decrease significantly with aging and cognitive decline and that lower expression levels correlate with declining cognitive ability, particularly in CA1 and CA3. These data suggest that decreased expression of NgR1-antagonizing proteins may exert a combinatorial effect with increased NgR1 signaling pathway components to result in abnormally strong suppression of synaptic plasticity in age-related cognitive impairment.