Aileen I. Pogue

Pathogenic microbes, the microbiome, and Alzheimer’s disease (AD)

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of cognitive and behavioral impairment in industrialized societies. The cause of AD is unknown and the major risk factor for AD is age. About 5% of all AD cases have a genetic or familial cause however the vast majority of all AD cases (~95%) are of sporadic origin. Both the familial and the sporadic forms of AD share a common disease phenotype involving at least eight characteristic features including (i) evidence of uncontrolled oxidative stress; (ii) up-regulated pro-inflammatory signaling; (iii) changes in innate-immune signaling; (iv) the progressive accumulation of lesions including neurofibrillary tangles (NFT) and amyloid beta (Aβ)-containing senile plaques (SP); (v) significant synaptic signaling deficits; (vi) neurite and brain cell atrophy; (vii) progressively altered gene expression patterns that are different from healthy brain aging; and (viii) progressive cognitive impairment and dementia in the host. There is currently no cure or adequate clinical treatment for AD, and it remains unclear how AD originates and propagates throughout the brain and central nervous system (CNS). Results from recent genome-wide association studies (GWAS) indicate that a significant portion of AD-relevant gene signals are not located within gene coding regions suggesting the contribution of epigenetic or environmental factors to AD risk. The potential contribution of pathogenic microbes to aging and AD is becoming increasingly recognized (Miklossy, 2011; Cho and Blaser, 2012; Bhattacharjee and Lukiw, 2013; Poole et al., 2013; Heintz and Mair, 2014; Huang et al., 2014; Mancuso et al., 2014). Importantly, most of the changes seen in AD, such as inflammation, brain cell atrophy, immunological aberrations, amyloidogenesis, altered gene expression and cognitive deficits are also seen as a consequence of microbial infection (Cho and Blaser, 2012; Yatsunenko et al., 2012; Bhattacharjee and Lukiw, 2013; Foster and McVey Neufeld, 2013; Kim et al., 2013; Heintz and Mair, 2014; Mancuso et al., 2014). This brief communication will review some recent observations on the potential contribution of pathogens to neurological dysfunction, with specific reference to AD wherever possible.

Towards the prevention of potential aluminum toxic effects and an effective treatment for Alzheimer’s disease

In 1991, treatment with low dose intramuscular desferrioxamine (DFO), a trivalent chelator that can remove excessive iron and/or aluminum from the body, was reported to slow the progression of Alzheimers disease (AD) by a factor of two. Twenty years later this promising trial has not been followed up and why this treatment worked still is not clear. In this critical interdisciplinary review, we provide an overview of the complexities of AD and involvement of metal ions, and revisit the neglected DFO trial. We discuss research done by us and others that is helping to explain involvement of metal ion catalyzed production of reactive oxygen species in the pathogenesis of AD, and emerging strategies for inhibition of metal-ion toxicity. Highlighted are insights to be considered in the quests to prevent potentially toxic effects of aluminum toxicity and prevention and intervention in AD.

Retinal amyloid peptides and complement factor H in transgenic models of Alzheimer's disease.

Murine transgenic models of Alzheimers disease (Tg-AD) have been useful to analyze the contribution of &bgr;-amyloid precursor protein (&bgr;APP), A&bgr;42 peptide deposition, and the proinflammatory mechanisms that characterize Alzheimer-type neuropathology. In this report, we have studied the levels of &bgr;APP, A&bgr;40 and A&bgr;42 peptide, as well as the innate immune and inflammatory response-regulator complement factor H in the brain and retina in four different Tg-AD models including Tg2576, PSAPP, 3xTg-AD, and 5xFAD. Aged, symptomatic 5xFAD mice showed the highest retinal abundance of A&bgr;42 peptides and the highest deficits in complement factor H. This may be a useful model to study the mechanisms of amyloid-mediated inflammatory degeneration. The superior colliculus and retina obtained from late-stage Alzheimers disease revealed upregulated amyloidogenic and inflammatory signaling along the anteroposterior axis of the retinal-primary visual cortex pathway.

Selective accumulation of aluminum in cerebral arteries in Alzheimer's disease (AD).

Once biologically available aluminum bypasses gastrointestinal and blood-brain barriers, this environmentally-abundant neurotoxin has an exceedingly high affinity for the large pyramidal neurons of the human brain hippocampus. This same anatomical region of the brain is also targeted by the earliest evidence of Alzheimers disease (AD) neuropathology. The mechanism for the selective targeting and transport of aluminum into the hippocampus of the human brain is not well understood. In an effort to improve our understanding of a pathological aluminum entry system into the brain, this study examined the aluminum content of 8 arteries that supply blood to the hippocampus, including the aorta and several cerebral arteries. In contrast to age-matched controls, in AD patients we found a gradient of increasing aluminum concentration from the aorta to the posterior cerebral artery that supplies blood to the hippocampus. Primary cultures of human brain endothelial cells were found to have an extremely high affinity for aluminum when compared to other types of brain cells. Together, these results suggest for the first time that endothelial cells that line the cerebral vasculature may have biochemical attributes conducive to binding and targeting aluminum to selective anatomical regions of the brain, such as the hippocampus, with potential downstream pro-inflammatory and pathogenic consequences.

miRNA-155 up-regulation and complement factor H (CFH) deficits in Down’s Syndrome

Down’s syndrome, a congenital disorder associated with cognitive impairment and early-onset Alzheimers disease, is a progressive genetic pathology resulting from full or partial triplication of chromosome 21. Down’s syndrome brain is typified by activated microglia, increases in inflammatory signaling, and an aberrant immune system. In these studies, a screening of micro-RNA (miRNA) from Down’s syndrome brain and peripheral tissues indicated an upregulation of a chromosome 21-encoded miRNA-155 and a decrease in the abundance of the miRNA-155 mRNA target complement factor H (CFH), an important repressor of the innate immune response. Stressed primary human neuronal–glial cells indicated both miRNA-155 increase and CFH downregulation, an effect that was reversed using anti-miRNA-155. These findings suggest that immunopathological deficits associated with Down’s syndrome can, in part, be explained by a generalized miRNA-155-mediated downregulation of CFH that may contribute to both brain and systemic immune pathology.

The Gastrointestinal Tract Microbiome and Potential Link to Alzheimer’s Disease

Accumulating clinical- and scientific research-based evidence is driving our increased awareness of the significance of the human microbiome (HM) to the healthy and homeostatic operation of the human central nervous system (CNS). HM communities occupy several different but distinct microbial ecosystems on and within the human body, including nasal, oral, and otic cavities, the surface of the skin and the urogenital and the gastrointestinal (GI) tracts. The complex symbiotic inter-relationship between the GI-tract microbiome and its host is strongly influenced by diet and nutrition, and when optimized can be highly beneficial to food digestion, nutrient intake, and immune health (1–6). For example, dietary composition ultimately affects the structure, organization, function, and speciation of the HM occupying the GI tract, in part by supplying multiple substrates for microbial metabolism. Typical Western diets containing high fat–cholesterol, low amounts of soluble and insoluble fiber, and sugar- and salt-enrichment not only impart deleterious nutrition but also dietary constraints on the HM. This in turn impacts the supply of microbiome-generated molecules absorbed into the systemic circulation for transport into the extensive neurovasculature of the CNS. This short communication will focus on emerging ideas concerning the contribution of the GI-tract microbiome to human neurological disease with emphasis on Alzheimer’s disease (AD) wherever possible.

MicroRNA (miRNA): Sequence and stability, viroid-like properties, and disease association in the CNS

MicroRNAs (miRNAs) constitute a relatively recently-discovered class of small non-coding RNAs (sncRNAs) that are gaining considerable attention in the molecular-genetic regulatory mechanisms that contribute to human health and disease. As highly soluble and mobile entities, emerging evidence indicates that miRNAs posess a highly selected ribonucleotide sequence structure, are part of an evolutionary ancient genetic signaling system, resemble the plant pathogens known as viroids in their structure, mode of generation and function, and are very abundant in the physiological fluids that surround cells and tissues. Persistence and altered abundance of miRNAs in the extracellular fluid (ECF) or cerebrospinal fluid (CSF) may play a role in the intercellular spreading of disease systemically, and throughout functionally-linked cellular and tissue systems such as the central nervous system (CNS). This short communication will review some of the more fascinating features of these highly structured single stranded RNAs (ssRNAs) with emphasis on their presence and function in the human CNS, with particular reference to Alzheimer׳s disease (AD) wherever possible.

Metal-Sulfate Induced Generation of ROS in Human Brain Cells: Detection Using an Isomeric Mixture of 5- and 6-Carboxy-2′,7′-Dichlorofluorescein Diacetate (Carboxy-DCFDA) as a Cell Permeant Tracer

Evolution of reactive oxygen species (ROS), generated during the patho-physiological stress of nervous tissue, has been implicated in the etiology of several progressive human neurological disorders including Alzheimer’s disease (AD) and amylotrophic lateral sclerosis (ALS). In this brief communication we used mixed isomers of 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (carboxy-DCFDA; C25H14Cl2O9; MW 529.3), a novel fluorescent indicator, to assess ROS generation within human neuronal-glial (HNG) cells in primary co-culture. We introduced pathological stress using the sulfates of 12 environmentally-, industrially- and agriculturally-relevant divalent and trivalent metals including Al, Cd, Cu, Fe, Hg, Ga, Mg, Mn, Ni, Pb, Sn and Zn. In this experimental test system, of all the metal sulfates analyzed, aluminum sulfate showed by far the greatest ability to induce intracellular ROS. These studies indicate the utility of using isomeric mixtures of carboxy-H2DCFDA diacetates as novel and highly sensitive, long-lasting, cell-permeant, fluorescein-based tracers for quantifying ROS generation in intact, metabolizing human brain cells, and in analyzing the potential epigenetic contribution of different metal sulfates to ROS-generation and ROS-mediated neurological dysfunction.

MicroRNA (miRNA) Signaling in the Human CNS in Sporadic Alzheimer’s Disease (AD)-Novel and Unique Pathological Features

Of the approximately ~2.65 × 103 mature microRNAs (miRNAs) so far identified in Homo sapiens, only a surprisingly small but select subset—about 35–40—are highly abundant in the human central nervous system (CNS). This fact alone underscores the extremely high selection pressure for the human CNS to utilize only specific ribonucleotide sequences contained within these single-stranded non-coding RNAs (ncRNAs) for productive miRNA–mRNA interactions and the down-regulation of gene expression. In this article we will: (i) consolidate some of our still evolving ideas concerning the role of miRNAs in the CNS in normal aging and in health, and in sporadic Alzheimer’s disease (AD) and related forms of chronic neurodegeneration; and (ii) highlight certain aspects of the most current work in this research field, with particular emphasis on the findings from our lab of a small pathogenic family of six inducible, pro-inflammatory, NF-κB-regulated miRNAs including miRNA-7, miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155. This group of six CNS-abundant miRNAs significantly up-regulated in sporadic AD are emerging as what appear to be key mechanistic contributors to the sporadic AD process and can explain much of the neuropathology of this common, age-related inflammatory neurodegeneration of the human CNS.

Pathogenic microRNAs Common to Brain and Retinal Degeneration; Recent Observations in Alzheimer’s Disease and Age-Related Macular Degeneration

MicroRNAs (miRNAs), ~22 nt single-stranded non-coding RNAs (ncRNAs) abundant in the human brain and retina, have emerged as significant post-transcriptional regulators of messenger RNA (mRNA) abundance and complexity in the human central nervous system (CNS) in aging, health, and disease. Of the 2050 different miRNAs in the human body so far identified, only about 25–30 are abundant in either the brain or the retina, underscoring the high selection pressure carried by RNA sequences located within these select ncRNAs (1–7). It is noteworthy to point out that: (i) that brain neocortex and retina share a common neuroectodermal origin; (ii) that brain and retina share a subfamily of specific miRNA species; and (iii) that the multilayered assemblies of both neural and retinal cells are targeted by pathogenic processes that drive progressive pro-inflammatory neurodegeneration (5–9). Indeed, pathologically up-regulated miRNAs common to both the prototypic age-related inflammatory degeneration of the brain in Alzheimer’s disease (AD) and of the retina in age-related macular degeneration (AMD) appear to be associated with deficits in the expression of messenger RNA (mRNA) and gene families involved in the innate-immune response, inflammation, neurotrophism, synaptogenesis, and amyloidogenesis (Figure ​(Figure1).1). In this “Opinion” paper for the Frontiers in Neurology Special Research Topic, we will highlight some of the most recent work in this research area, with emphasis on a family of five up-regulated pro-inflammatory miRNAs – miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155 – that are emerging as key mechanistic contributors to the AD and AMD process. Figure 1 (A) Color-coded cluster analysis of significantly up-regulated microRNAs (miRNAs) in the neocortex of AD (N = 5) versus age-matched controls (N = 5) and (B) in the whole retina of AMD (N = 5) versus age-matched ... Homeostatic levels of specific miRNAs are natural indicators of neurological health of both the brain and retina (2–10, 31). Recently, multiple independent neurological research laboratories have provided evidence for the up-regulation of a small group of five inducible miRNAs in age-related diseases involving a progressive inflammatory degeneration. That these five miRNAs – miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155 – are up-regulated in both AD and AMD underscores the concept that the brain and retina share common pathological signaling of a pre-existing subfamily of miRNAs that individually contribute to various aspects of neurodegenerative disease (5–12, 31–35). Accumulating evidence, including very recent research findings over the last 6 months indicate that each of these miRNAs share the following six features: (i) that they are basally expressed in control brain neocortex and retina (2–9); (ii) that in vitro they can be induced by a wide range of environmental- and inflammation-linked physiological stressors, including pro-inflammatory cytokines, amyloid beta (Aβ42) peptides, neurotoxic metal sulfates, and neurotropic viruses such as herpes simplex virus-1 (HSV-1) (12, 16, 17, 32–35); (iii) that this group of five pro-inflammatory miRNAs are over-expressed at least twofold in stressed brain or retinal cells and in AD or AMD affected tissues (14, 15, 32); (iv) that together, via down-regulation of multiple mRNA targets (and hence deficits in the expression of genes encoded by those mRNAs) they regulate various pathophysiological features characteristic of AD and AMD, including impairments in phagocytosis, synaptogenesis, neurotrophism, NF-kB signaling and stimulation of progressive inflammation and amyloidogenesis (Figure ​(Figure1)1) (7, 12, 13, 23, 26–28, 36); (v) that all five of these pro-inflammatory miRNAs are under transcriptional control by NF-kB (chiefly the heterotypic p50/p65 dimer) in human primary neuronal-glial co-cultures, AD and AMD tissues (7, 11–13, 23, 26–28, 36, 37); and (vi) that both NF-kB inhibitors and anti-microRNAs (anti-miRs) effectively knock down their expression in human brain and retinal cell culture experiments, and may ultimately be of use therapeutically in the clinical management of AD or AMD (17, 18, 26–29). Much of the recent research work emphasizing this commonality of the same miRNAs in basic pathological processes involving brain and retinal degeneration, as exemplified by miRNA profiling in AD, AMD, and transgenic AD or AMD (TgAD, TgAMD) models, has been summarized in Figure ​Figure11 (5–10, 12, 14, 17, 25, 31–35). First, when compared to the unchanging 22 nt miRNA-183 and the 120 nt 5S ribosomal RNA (5S rRNA; 5SRNA) control markers, the five member pro-inflammatory microRNAs miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155 are found to be amongst the most consistently up-regulated miRNAs in both degenerating human brain neocortex (Figure ​(Figure1A)1A) and retina (Figure ​(Figure1B).1B). Of this group of five pro-inflammatory microRNAs, miRNA-146a and miRNA-155 are typically found to be increased ~2.5- to 3.3-fold over age-matched controls (Figure ​(Figure1C).1C). To add another layer of genetic complexity for post-transcriptional regulation, both miRNA-146a and miRNA-155 recognize an overlapping 3′ untranslated region (3′UTR) of the complement factor H (CFH) mRNA (highlighted in green; CFH loss-of-function mutations or CFH expression deficits are associated with both AD and AMD; see below; Figure ​Figure1D).1D). Indeed, the up-regulation of these same five pro-inflammatory miRNAs (yellow ovals in Figure ​Figure1E)1E) appear to form a highly interactive miRNA–mRNA network that can in part explain the down-regulation of specific brain and retinal genes (black rectangles) involved in phagocytosis, inflammation, synaptogenesis, neurotrophism, NF-kB signaling, and amyloidogenesis (Figure ​(Figure1E;1E; see also the legend to Figure ​Figure11 wherein the details of this highly interactive network are further described). Alterations in innate-immune signaling are a consistent feature of both AD and AMD (4, 5, 9, 15). The highly soluble, hydrophilic 155-kDa glycoprotein CFH is one very illustrative example of an innate-immune repressor and complement control protein whose abundance and/or activity is significantly down-regulated in both AD and AMD [(9, 15, 21, 22, 35); see Figure ​Figure1D].1D]. CFH (chr 1q32; also known as AC3bINA, adrenomedullin binding protein-1, AM binding protein-1 factor H, β1H globulin, H factor, and H factor-1) is an important member of the regulator of complement activation (RCA) group of proteins encoded within the RCA gene cluster and normally performs a systemic sentinel function against unscheduled or spontaneous immune system activation (9, 15). CFH mRNA abundance is down-regulated in AD and/or AMD by a miRNA-146a- and/or miRNA-155–CFH–3′UTR-based complementarity mechanism and/or by a Y402H loss-of-function mutation (15, 21, 22). Hence an insufficiency in a homeostatic amount of functioning CFH (as down-regulated by miRNA-146a and miRNA-155) may have the same end result as the loss-of-function Y402H mutation in CFH (21, 22). It is important to note that CFH mRNA and hence CFH gene expression appears to be down-regulated by at least two different miRNAs – miRNA-146a and/or miRNA-155 – and their differential recognition of overlapping binding sites in the human CFH mRNA 3′UTR may be dependent on yet-to-be-defined genetic factors and mechanisms characteristic of individual brain or retinal cells [Figure ​[Figure1D;1D; (9, 15, 21, 22, 35)]. In summary, it is our opinion that in miRNA research in human degenerative diseases including AD and AMD, several critical concerns have surfaced: (i) that brain and retinal miRNAs typically possess limited stabilities, however miRNA half-lives can be considerably extended via their sequestration into exosomes or the use of other protective strategies such as adsorption or tertiary folding into RNAse-resistant structures that may escape initial miRNA detection using traditional methods (17, 18, 23–25); (ii) that accurate quantification of miRNAs is technically feasible although it still remains challenging due to the small size of mature miRNA isoforms, adsorption to “inert” surfaces, high sequence homology amongst individual miRNAs, 5′ and 3′ end polymorphisms, spatial-temporal expression patterns and high dynamic range of miRNA expression (13, 17, 18, 24); (iii) that miRNA profiling in different AD or AMD studies suffers from a poor consensus regarding their abundance and complexity; the latter a very recently acknowledged concern in the field (4–7, 14, 17); and (iv) discrepancies of miRNA abundances in anatomical areas sampled, variations in patient drug history, the PMI of the AD and AMD patients and other factors. Together these constitute practical methodological challenges, especially in the realm of useful biomarkers and diagnostics for AD or AMD detection (3, 6, 17, 25, 34). Despite these recent concerns data has begun to filter through on the involvement of distinct miRNA families and miRNA–mRNA signaling networks linked to innate-immune system alterations, inflammatory, neurotrophic, and amyloidogenic consequences in AD and AMD. These have steadily yielded a deeper appreciation into the onset and propagation of complex miRNA–mRNA-modulated biological networks that directly underlie the pathogenesis of AD and AMD. Lastly, miRNAs are highly soluble and mobile, and are able to transverse plasma membranes either freely, adsorbed to carrier molecules or contained within exosomes (17, 19, 23, 25). That AD and AMD are both progressive “propagating” disease entities suggest a potential “spreading factor” role for selective miRNAs in the cognitive and visual circuitry, an evolving research area in which specific combinations of miRNAs may be playing hitherto unrecognized pathogenic roles.