Todd E. Morgan

Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of β-secretase

Recent efforts have revealed that numerous protein-coding messenger RNAs have natural antisense transcript partners, most of which seem to be noncoding RNAs. Here we identify a conserved noncoding antisense transcript for β-secretase-1 (BACE1), a crucial enzyme in Alzheimers disease pathophysiology. The BACE1-antisense transcript (BACE1-AS) regulates BACE1 mRNA and subsequently BACE1 protein expression in vitro and in vivo. Upon exposure to various cell stressors including amyloid-β 1–42 (Aβ 1–42), expression of BACE1-AS becomes elevated, increasing BACE1 mRNA stability and generating additional Aβ 1–42 through a post-transcriptional feed-forward mechanism. BACE1-AS concentrations were elevated in subjects with Alzheimers disease and in amyloid precursor protein transgenic mice. These data show that BACE1 mRNA expression is under the control of a regulatory noncoding RNA that may drive Alzheimers disease–associated pathophysiology. In summary, we report that a long noncoding RNA is directly implicated in the increased abundance of Aβ 1–42 in Alzheimers disease.

Progesterone Receptors: Form and Function in Brain

Emerging data indicate that progesterone has multiple non-reproductive functions in the central nervous system to regulate cognition, mood, inflammation, mitochondrial function, neurogenesis and regeneration, myelination and recovery from traumatic brain injury. Progesterone-regulated neural responses are mediated by an array of progesterone receptors (PR) that include the classic nuclear PRA and PRB receptors and splice variants of each, the seven transmembrane domain 7TMPRbeta and the membrane-associated 25-Dx PR (PGRMC1). These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. Remarkably, PRs are broadly expressed throughout the brain and can be detected in every neural cell type. The distribution of PRs beyond hypothalamic borders, suggests a much broader role of progesterone in regulating neural function. Despite the large body of evidence regarding progesterone regulation of reproductive behaviors and estrogen-inducible responses as well as effects of progesterone metabolite neurosteroids, much remains to be discovered regarding the functional outcomes resulting from activation of the complex array of PRs in brain by gonadally and/or glial derived progesterone. Moreover, the impact of clinically used progestogens and developing selective PR modulators for targeted outcomes in brain is a critical avenue of investigation as the non-reproductive functions of PRs have far-reaching implications for hormone therapy to maintain neurological health and function throughout menopausal aging.

Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies

In recent studies of transgenic models of Alzheimers disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1–42 (Aβ1−42) solutions (mixtures of Aβ monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non‐fibrillar forms of Aβ. It is now known that Aβ toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Aβ1−42 oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Aβ1−42 solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Aβ in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell‐attached oligomers to receptor‐like puncta, and for immunoblots that show the presence of SDS‐stable oligomers in Alzheimers brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno‐neutralization of soluble Aβ‐derived toxins. Analogous immuno‐neutralization of oligomers in humans may be a key in AD vaccines.

Caloric restriction attenuates Aβ-deposition in Alzheimer transgenic models

Dietary influences on Alzheimer disease (AD) are gaining recognition. Because many aging processes are attenuated in laboratory mammals by caloric restriction (CR), we examined the effects of short-term CR in two AD-transgenic mice, APP(swe/ind) (J20) and APP(swe) + PS1(M146L) (APP + PS1). CR substantially decreased the accumulation of Abeta-plaques in both lines: by 40% in APP(swe/ind) (CR, 6 weeks), and by 55% in APP + PS1 (CR, 14 weeks). CR also decreased astrocytic activation (GFAP immunoreactivity). These influences of CR on AD-transgenic mice are consistent with epidemiological reports that show that high caloric diets associate with the risk of AD, and suggest that dietary interventions in adult life might slow disease progression.

Astrocytes and Microglia Respond to Estrogen with Increased apoE mRNAin Vivoandin Vitro

This study examined the regulation of apolipoprotein E (apoE) by 17beta-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as an in vivo model and cultured astrocytes and mixed glia as in vitro models. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA. In vitro, E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens. In situ hybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microglia in vivo and the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimers disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.

Joint Genome-Wide Profiling of miRNA and mRNA Expression in Alzheimer's Disease Cortex Reveals Altered miRNA Regulation

Although microRNAs are being extensively studied for their involvement in cancer and development, little is known about their roles in Alzheimers disease (AD). In this study, we used microarrays for the first joint profiling and analysis of miRNAs and mRNAs expression in brain cortex from AD and age-matched control subjects. These data provided the unique opportunity to study the relationship between miRNA and mRNA expression in normal and AD brains. Using a non-parametric analysis, we showed that the levels of many miRNAs can be either positively or negatively correlated with those of their target mRNAs. Comparative analysis with independent cancer datasets showed that such miRNA-mRNA expression correlations are not static, but rather context-dependent. Subsequently, we identified a large set of miRNA-mRNA associations that are changed in AD versus control, highlighting AD-specific changes in the miRNA regulatory system. Our results demonstrate a robust relationship between the levels of miRNAs and those of their targets in the brain. This has implications in the study of the molecular pathology of AD, as well as miRNA biology in general.

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease.

The DSCR1(Adapt78) gene was independently discovered as a resident of the “Down syndrome candidate region”and as an “adaptive response”shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized thatDSCR1 (Adapt78) might also be involved in the development of Alzheimers disease. To address this question we first studiedDSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimers disease patients and individuals who had died with no Alzheimers diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimers patients as in controls.DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimers disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimers symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid β1–42 peptide, which is a major component of senile plaques in Alzheimers, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimers disease as amyloid protein, senile plaques, and neurofibrillary tangles.

TGF-β1 mRNA Increases in Macrophage/Microglial Cells of the Hippocampus in Response to Deafferentation and Kainic Acid-Induced Neurodegeneration

This study examined TGF-beta 1 mRNA levels and cellular localization in the F344 rat hippocampus following deafferentation or kainic acid (KA)-induced neurodegeneration. By RNA solution hybridization, TGF-beta 1 transcripts were at low prevalence in intact adult rat hippocampus (0.02 pg/microgram total RNA). Four days after unilateral entorhinal cortex lesioning (ECL), TGF-beta 1 mRNA increased threefold in the ipsilateral hippocampus. This increase was localized to the outer molecular layer of the dentate gyrus, where gliosis, synapse loss, and synaptic reorganization occur. TGF-beta 1 mRNA also increased in the hippocampus after KA-induced limbic seizures, particularly in the areas of the hippocampus undergoing neurodegeneration. Microglia [OX-42 immunoreactive (IR) cells] responded to these two lesions with distinct morphological changes. Combined immunocytochemistry-in situ hybridization showed that TGF-beta 1 mRNA was localized to reactive microglia (OX-42-IR, with blunt processes), but not to resting ramified microglia (OX-42-IR, with numerous fine processes) or to astrocytes (GFAP-IR). After ECL, round macrophage-like cells (OX-42-IR with TGF-beta 1 mRNA) were seen at the wound site. Thus, brain macrophage/microglial cells produce TGF-beta 1 mRNA in the hippocampus in response to deafferentation and neurodegeneration.

The mosaic of brain glial hyperactivity during normal ageing and its attenuation by food restriction.

Food restriction of adult rodents increases lifespan, with commensurate attenuation of age-related pathological lesions in many organs, as well as attenuation of normal ageing changes that are distinct from gross lesions. Previous work showed that chronic food restriction attenuated age-associated astrocyte and microglial hyperactivity in the hippocampal hilus, as measured by expression of glial fibrillary acidic protein and major histocompatibility complex II antigen (OX6). Here, we examined other markers of astrocyte and microglial activation in gray and white matter regions of ad libitum-fed (Brown Norway x Fischer 344) F1 male rats aged three and 24 months and chronic food-restricted rats aged 24 months. In situ hybridization and immunohistochemical techniques evaluated glial expression of glial fibrillary acidic protein, apolipoprotein E, apolipoprotein J (clusterin), heme oxygenase-1, complement 3 receptor (OX42), OX6 and transforming growth factor-beta1. All markers were elevated in the corpus callosum during ageing and were attenuated by food restriction, but other regions showed marked dissociation of the extent and direction of changes. Astrocytic activation, as measured with glial fibrillary acidic protein expression (coding and intron-containing RNA, immunoreactivity), increased with age in the corpus callosum, basal ganglia and hippocampus. Generally, food restriction attenuated the age-related increase in glial fibrillary acidic protein messenger RNA and immunoreactivity. Food restriction also reduced the age-related increase in apolipoprotein J and E messenger RNA and heme oxygenase-1 immunoreactivity in the basal ganglia and corpus callosum. However, astrocytes in the hilus of the hippocampus showed an age-related decrease in apolipoprotein J and E messenger RNA, which was further intensified by food restriction. The age-associated microglial activation measured by OX6 and OX42 immunoreactivity was reduced by food restriction in most subregions. The localized subsets of glial age changes and effects of food restriction comprise a mosaic of ageing consistent with the regional heterogeneity of ageing changes reported by others. In particular, age has a differential effect on astrocytic and microglial hyperactivity in gray versus white matter areas. The evident mosaic of glial ageing and responses to food restriction suggests that multiple mechanisms are at work during ageing.

Functional annotation and network reconstruction through cross-platform integration of microarray data

The rapid accumulation of microarray data translates into a need for methods to effectively integrate data generated with different platforms. Here we introduce an approach, 2nd-order expression analysis, that addresses this challenge by first extracting expression patterns as meta-information from each data set (1st-order expression analysis) and then analyzing them across multiple data sets. Using yeast as a model system, we demonstrate two distinct advantages of our approach: we can identify genes of the same function yet without coexpression patterns and we can elucidate the cooperativities between transcription factors for regulatory network reconstruction by overcoming a key obstacle, namely the quantification of activities of transcription factors. Experiments reported in the literature and performed in our lab support a significant number of our predictions.