Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).

Early-life DNA methylation profiles are indicative of age-related transcriptome changes.

BACKGROUND: Alterations to cellular and molecular programs with brain aging result in cognitive impairment and susceptibility to neurodegenerative disease. Changes in DNA methylation patterns, an epigenetic modification required for various CNS functions are observed with brain aging and can be prevented by anti-aging interventions, but the relationship of altered methylation to gene expression is poorly understood. RESULTS: Paired analysis of the hippocampal methylome and transcriptome with aging of male and female mice demonstrates that age-related differences in methylation and gene expression are anti-correlated within gene bodies and enhancers. Altered promoter methylation with aging was found to be generally un-related to altered gene expression. A more striking relationship was found between methylation levels at young age and differential gene expression with aging. Highly methylated gene bodies and promoters in early life were associated with age-related increases in gene expression even in the absence of significant methylation changes with aging. As well, low levels of methylation in early life were correlated to decreased expression with aging. This relationship was also observed in genes altered in two mouse Alzheimer's models. CONCLUSION: DNA methylation patterns established in youth, in combination with other epigenetic marks, were able to accurately predict changes in transcript trajectories with aging. These findings are consistent with the developmental origins of disease hypothesis and indicate that epigenetic variability in early life may explain differences in aging trajectories and age-related disease.

Exposure to environmental enrichment attenuates addiction-like behavior and alters molecular effects of heroin self-administration in rats.

Environmental factors profoundly affect the addictive potential of drugs of abuse and may also modulate the neuro-anatomical/neuro-chemical impacts of uncontrolled drug use and relapse propensity. This study examined the impact of environmental enrichment on heroin self-administration, addiction-related behaviors, and molecular processes proposed to underlie these behaviors. Male Sprague-Dawley rats in standard and enriched housing conditions intravenously self-administered similar amounts of heroin over 14 days. However, environmental enrichment attenuated progressive ratio, extinction, and reinstatement session responding after 14 days of enforced abstinence. Molecular mechanisms, namely DNA methylation and gene expression, are proposed to underlie abstinence-persistent behaviors. A global reduction in methylation is reported to coincide with addiction, but no differences in total genomic methylation or repeat element methylation were observed in CpG or non-CpG (CH) contexts across the mesolimbic circuitry as assessed by multiple methods including whole genome bisulfite sequencing. Immediate early gene expression associated with drug seeking, taking, and abstinence also were examined. EGR1 and EGR2 were suppressed in mesolimbic regions with heroin-taking and environmental enrichment. Site-specific methylation analysis of EGR1 and EGR2 promoter regions using bisulfite amplicon sequencing (BSAS) revealed hypo-methylation in the EGR2 promoter region and EGR1 intragenic CpG sites with heroin-taking and environmental enrichment that was associated with decreased mRNA expression. Taken together, these findings illuminate the impact of drug taking and environment on the epigenome in a locus and gene-specific manner and highlight the need for positive, alternative rewards in the treatment and prevention of drug addiction.

Caloric restriction mitigates age-associated hippocampal differential CG and non-CG methylation.

Brain aging is marked by cognitive decline and susceptibility to neurodegeneration. Calorie restriction (CR) increases neurogenesis, improves memory function, and protects from age-associated neurological disorders. Epigenetic mechanisms, including DNA methylation, are vital to normal central nervous system cellular and memory functions and are dysregulated with aging. The beneficial effects of CR have been proposed to work through epigenetic processes, but this is largely unexplored. We therefore tested whether life long CR prevents age-related hippocampal DNA methylation changes. Hippocampal DNA from young (3 months) and old (24 months) male mice fed ad libitum and 24-month-old mice fed a 40% calorie-restricted diet from 3 months of age were examined by genome-wide bisulfite sequencing to measure methylation with base specificity. Over 27 million CG and CH (non-CG) sites were examined. Of the ∼40,000 differentially methylated CG and ∼80,000 CH sites with aging, >1/3 were prevented by CR and were found across genomic regulatory regions and gene pathways. CR also caused alterations to CG and CH methylation at sites not differentially methylated with aging, and these CR-specific changes demonstrated a different pattern of regulatory element and gene pathway enrichment than those affected by aging. CR-specific DNA methyltransferase 1 and Tet methylcytosine dioxygenase 3 promoter hypermethylation corresponded to reduced gene expression. These findings demonstrate that CR attenuates age-related CG and CH hippocampal methylation changes, in combination with CR-specific methylation that may also contribute to the neuroprotective effects of CR. The prevention of age-related methylation alterations is also consistent with the prolongevity effects of CR working through an epigenetic mechanism.

Insulin-like growth factor receptor signaling regulates working memory, mitochondrial metabolism, and amyloid-ß uptake in astrocytes.

OBJECTIVE: A decline in mitochondrial function and biogenesis as well as increased reactive oxygen species (ROS) are important determinants of aging. With advancing age, there is a concomitant reduction in circulating levels of insulin-like growth factor-1 (IGF-1) that is closely associated with neuronal aging and neurodegeneration. In this study, we investigated the effect of the decline in IGF-1 signaling with age on astrocyte mitochondrial metabolism and astrocyte function and its association with learning and memory. METHODS: Learning and memory was assessed using the radial arm water maze in young and old mice as well as tamoxifen-inducible astrocyte-specific knockout of IGFR (GFAP-CreTAM/igfrf/f). The impact of IGF-1 signaling on mitochondrial function was evaluated using primary astrocyte cultures from igfrf/f mice using AAV-Cre mediated knockdown using Oroboros respirometry and Seahorse assays. RESULTS: Our results indicate that a reduction in IGF-1 receptor (IGFR) expression with age is associated with decline in hippocampal-dependent learning and increased gliosis. Astrocyte-specific knockout of IGFR also induced impairments in working memory. Using primary astrocyte cultures, we show that reducing IGF-1 signaling via a 30-50% reduction IGFR expression, comparable to the physiological changes in IGF-1 that occur with age, significantly impaired ATP synthesis. IGFR deficient astrocytes also displayed altered mitochondrial structure and function and increased mitochondrial ROS production associated with the induction of an antioxidant response. However, IGFR deficient astrocytes were more sensitive to H2O2-induced cytotoxicity. Moreover, IGFR deficient astrocytes also showed significantly impaired glucose and Aß uptake, both critical functions of astrocytes in the brain. CONCLUSIONS: Regulation of astrocytic mitochondrial function and redox status by IGF-1 is essential to maintain astrocytic function and coordinate hippocampal-dependent spatial learning. Age-related astrocytic dysfunction caused by diminished IGF-1 signaling may contribute to the pathogenesis of Alzheimer's disease and other age-associated cognitive pathologies.

Revisiting the genomic hypomethylation hypothesis of aging.

The genomic hypomethylation hypothesis of aging proposes that an overall decrease in global DNA methylation occurs with age, and it has been argued that the decrease in global DNA methylation could be an important factor in aging, resulting in the relaxation of gene expression regulation and abnormal gene expression. Since it was initially observed that DNA methylation decreased with age in 1974, 16 articles have been published describing the effect of age on global DNA methylation in various tissues from rodents and humans. We critically reviewed the publications on the effect of age on DNA methylation and the expression of the enzymes involved in DNA methylation to evaluate the validity of the hypomethylation hypothesis of aging. On the basis of the current scientific literature, we conclude that a decrease in the global methylation of the genome occurs in most if not all tissues/cells as an animal ages. However, age-related changes in DNA methylation in specific regions or at specific sites in the genome occur even though the global DNA methylation does not change.

Analysis of DNA modifications in aging research.

As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through next-generation sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-the-art for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

Retinal gene expression responses to aging are sexually divergent.

PURPOSE: Sex and age are critical factors in a variety of retinal diseases but have garnered little attention in preclinical models. The current lack of knowledge impairs informed decision making regarding inclusion and design of studies that incorporate both sexes and/or the effects of aging. The goal of this study was to examine normative mouse retina gene expression in both sexes and with advancing age. METHODS: Retinal gene expression in female and male C57BL/6JN mice at 3 months and 24 months of age were compared for sex differences and aging responses through whole transcriptome microarray analysis. Sex differences and age-related changes were examined in the context of cellular pathways and processes, regulatory patterns, and cellular origin, as well as for overlap with described changes in retinal disease models. Selected age and sex differences were confirmed with quantitative PCR. RESULTS: Age-related gene expression changes demonstrated commonalities and sexually divergent responses. Several cellular pathways and processes, especially inflammation-related, are affected and were over-represented in fibroblast, microglial, and ganglion cell-specific genes. Lifelong, and age-dependent, sex differences were observed and were over-represented in fibroblast-specific genes. Age and sex differences were also observed to be regulated in models of diabetic retinopathy, glaucoma, and other diseases. CONCLUSIONS: These findings demonstrate that most age-related changes in retinal gene expression are sexually divergent and that there are significant sex differences in gene expression throughout the lifespan. These data serve as a resource for vision researchers seeking to include sex and age as factors in their preclinical studies.

Sexually divergent DNA methylation patterns with hippocampal aging.

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age-related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome-wide analyses of age-related methylation changes have considered the factor of sex in a controlled animal model. High-depth, genome-wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non-CG (CH) contexts demonstrated age-related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under-represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex-specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome-wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.

Functional changes in the neural retina occur in the absence of mitochondrial dysfunction in a rodent model of diabetic retinopathy.

Diabetic retinopathy is a neurovascular diabetes complication resulting in vision loss. A wealth of literature reports retinal molecular changes indicative of neural deficits, inflammation, and vascular leakage with chronic diabetes, but the mechanistic causes of disease initiation and progression are unknown. Microvascular mitochondrial DNA (mtDNA) damage leading to mitochondrial dysfunction has been proposed to drive vascular dysfunction in retinopathy. However, growing evidence suggests that neural retina dysfunction precedes and may cause vascular damage. Therefore, we tested the hypothesis that neural mtDNA damage and mitochondrial dysfunction are an early initiating factor of neural diabetic retinopathy development in a rat streptozotocin-induced, Type I diabetes model. Mitochondrial function (oxygen consumption rates) was quantified in retinal synaptic terminals from diabetic and non-diabetic rats with paired retinal structural and function assessment (optical coherence tomography and electroretinography, respectively). Mitochondrial genome damage was assessed by identifying mutations and deletions across the mtDNA genome by high depth sequencing and absolute mtDNA copy number counting through digital PCR. Mitochondrial protein expression was assessed by targeted mass spectrometry. Retinal functional deficits and neural anatomical changes were present after 3 months of diabetes and prevented/normalized by insulin treatment. No marked dysfunction of mitochondrial activity, maladaptive changes in mitochondrial protein expression, alterations in mtDNA copy number, or increase in mtDNA damage was observed in conjunction with retinal functional and anatomical changes. These results demonstrate that neural retinal dysfunction with diabetes begins prior to mtDNA damage and dysfunction, and therefore retinal neurodegeneration initiation with diabetes occurs through other, non-mitochondrial DNA damage, mechanisms.

Sexually divergent induction of microglial-associated neuroinflammation with hippocampal aging.

BACKGROUND: The necessity of including both males and females in molecular neuroscience research is now well understood. However, there is relatively limited basic biological data on brain sex differences across the lifespan despite the differences in age-related neurological dysfunction and disease between males and females. METHODS: Whole genome gene expression of young (3 months), adult (12 months), and old (24 months) male and female C57BL6 mice hippocampus was analyzed. Subsequent bioinformatic analyses and confirmations of age-related changes and sex differences in hippocampal gene and protein expression were performed. RESULTS: Males and females demonstrate both common expression changes with aging and marked sex differences in the nature and magnitude of the aging responses. Age-related hippocampal induction of neuroinflammatory gene expression was sexually divergent and enriched for microglia-specific genes such as complement pathway components. Sexually divergent C1q protein expression was confirmed by immunoblotting and immunohistochemistry. Similar patterns of cortical sexually divergent gene expression were also evident. Additionally, inter-animal gene expression variability increased with aging in males, but not females. CONCLUSIONS: These findings demonstrate sexually divergent neuroinflammation with aging that may contribute to sex differences in age-related neurological diseases such as stroke and Alzheimer's, specifically in the complement system. The increased expression variability in males suggests a loss of fidelity in gene expression regulation with aging. These findings reveal a central role of sex in the transcriptomic response of the hippocampus to aging that warrants further, in depth, investigations.

Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (Nuclear), P2 (Cytoskeletal), P3 (Neurosynaptosomal), PSD (Post-synaptic Densities), and SV (Synaptic Vesicle).

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, ß2M, Lilrb3, Klra2, CD247) display markedly different expression profiles across the hippocampus, cortex, cerebellum, brainstem, and retina. MHCI components, receptors, associated inflammatory transcripts (IL1α, IL1ß, IL6, TNFα), 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.

Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice.

BACKGROUND: Changes to the epigenome with aging, and DNA modifications in particular, have been proposed as a central regulator of the aging process, a predictor of mortality, and a contributor to the pathogenesis of age-related diseases. In the central nervous system, control of learning and memory, neurogenesis, and plasticity require changes in cytosine methylation and hydroxymethylation. Although genome-wide decreases in methylation with aging are often reported as scientific dogma, primary research reports describe decreases, increases, or lack of change in methylation and hydroxymethylation and their principle regulators, DNA methyltransferases and ten-eleven translocation dioxygenases in the hippocampus. Furthermore, existing data are limited to only male animals. RESULTS: Through examination of the hippocampus in young, adult, and old male and female mice by antibody-based, pyrosequencing, and whole-genome oxidative bisulfite sequencing methods, we provide compelling evidence that contradicts the genomic hypomethylation theory of aging. We also demonstrate that expression of DNA methyltransferases and ten-eleven translocation dioxygenases is not differentially regulated with aging or between the sexes, including the proposed cognitive aging regulator DNMT3a2. Using oxidative bisulfite sequencing that discriminates methylation from hydroxymethylation and by cytosine (CG and non-CG) context, we observe sex differences in average CG methylation and hydroxymethylation of the X chromosome, and small age-related differences in hydroxymethylation of CG island shores and shelves, and methylation of promoter regions. CONCLUSION: These findings clarify a long-standing misconception of the epigenomic response to aging and demonstrate the need for studies of base-specific methylation and hydroxymethylation with aging in both sexes.

Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle.

Mitochondria contain multiple copies of the circular mitochondrial genome (mtDNA) that encodes ribosomal RNAs and proteins locally translated for oxidative phosphorylation. Loss of mtDNA integrity, both altered copy number and increased mutations, is implicated in cellular dysfunction with aging. Published data on mtDNA copy number and aging is discordant which may be due to methodological limitations for quantifying mtDNA copy number. Existing quantitative PCR (qPCR) mtDNA copy number quantification methods provide only relative abundances and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA for normalization. We have developed and validated a novel absolute mtDNA copy number quantitation method that uses chip-based digital polymerase chain reaction (dPCR) to count the number of copies of mtDNA and used this novel method to assess the literature discrepancy in which there is no clear consensus whether mtDNA numbers change with aging in skeletal muscle. Skeletal muscle in old mice was found to have increased absolute mtDNA numbers compared to young controls. Furthermore, young Sod1 -/- mice were assessed and show an age-mimicking increase in skeletal muscle mtDNA. These findings reproduce a number of previous studies that demonstrate age-related increases in mtDNA. This simple and cost effective dPCR approach should enable precise and accurate mtDNA copy number quantitation in mitochondrial studies, eliminating contradictory studies of mitochondrial DNA content with aging.

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

Assessment of individual differences in the rat nucleus accumbens transcriptome following taste-heroin extended access.

Heroin addiction is a disease of chronic relapse that harms the individual through devaluation of personal responsibilities in favor of finding and using drugs. Only some recreational heroin users devolve into addiction but the basis of these individual differences is not known. We have shown in rats that avoidance of a heroin-paired taste cue reliably identifies individual animals with greater addiction-like behavior for heroin. Here rats received 5min access to a 0.15% saccharin solution followed by the opportunity to self-administer either saline or heroin for 6h. Large Suppressors of the heroin-paired taste cue displayed increased drug escalation, motivation for drug, and drug loading behavior compared with Small Suppressors. Little is known about the molecular mechanisms of these individual differences in addiction-like behavior. We examined the individual differences in mRNA expression in the nucleus accumbens (NAc) of rats that were behaviorally stratified by addiction-like behavior using next-generation sequencing. We hypothesized that based on the avoidance of the drug-paired cue there will be a unique mRNA profile in the NAc. Analysis of strand-specific whole genome RNA-Seq data revealed a number of genes differentially regulated in NAc based on the suppression of the natural saccharine reward. Large Suppressors exhibited a unique mRNA prolife compared to Saline controls and Small Suppressors. Genes related to immunity, neuronal activity, and behavior were differentially expressed among the 3 groups. In total, individual differences in avoidance of a heroin-paired taste cue are associated with addiction-like behavior along with differential NAc gene expression.

Targeted DNA methylation analysis by next-generation sequencing.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

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.

Insulin treatment normalizes retinal neuroinflammation but not markers of synapse loss in diabetic rats.

Diabetic retinopathy is one of the leading causes of blindness in developed countries, and a majority of patients with type I and type II diabetes will develop some degree of vision loss despite blood glucose control regimens. The effects of different insulin therapy regimens on early metabolic, inflammatory and neuronal retinal disease processes such as retinal neuroinflammation and synapse loss have not been extensively investigated. This study compared 3 months non-diabetic and streptozotocin (STZ)-induced diabetic Sprague Dawley rats. Diabetic rats received either no insulin treatment, systemic insulin treatment beginning after 1 week uncontrolled diabetes (early intervention, 11 weeks on insulin), or after 1.5 months uncontrolled diabetes (late intervention, 6 weeks on insulin). Changes in both whole animal metabolic and retinal inflammatory markers were prevented by early initiation of insulin treatment. These metabolic and inflammatory changes were also normalized by the later insulin intervention. Insulin treatment begun 1 week after diabetes induction ameliorated loss of retinal synapse markers. Synapse markers and presumably synapse numbers were equivalent in uncontrolled diabetes and when insulin treatment began at 1.5 months of diabetes. These findings are in agreement with previous demonstrations that retinal synapses are lost within 1 month of uncontrolled diabetes and suggest that synapses are not regained with glycemic control and restoration of insulin signaling. However, increased expression of metabolic and inflammatory markers associated with diabetes was reversed in both groups of insulin treatment. This study also emphasizes the need for insulin treatment groups in diabetic retinopathy studies to provide a more faithful modeling of the human condition.