Surjyadipta Bhattacharjee

Differential Regulation of Interleukin-1 Receptor-associated Kinase-1 (IRAK-1) and IRAK-2 by MicroRNA-146a and NF-κB in Stressed Human Astroglial Cells and in Alzheimer Disease

Specific microRNAs (miRNAs), small non-coding RNAs that support homeostatic gene expression, are significantly altered in abundance in human neurological disorders. In monocytes, increased expression of an NF-κB-regulated miRNA-146a down-regulates expression of the interleukin-1 receptor-associated kinase-1 (IRAK-1), an essential component of Toll-like/IL-1 receptor signaling. Here we extend those observations to the hippocampus and neocortex of Alzheimer disease (AD) brain and to stressed human astroglial (HAG) cells in primary culture. In 66 control and AD samples we note a significant up-regulation of miRNA-146a coupled to down-regulation of IRAK-1 and a compensatory up-regulation of IRAK-2. Using miRNA-146a-, IRAK-1-, or IRAK-2 promoter-luciferase reporter constructs, we observe decreases in IRAK-1 and increases in miRNA-146a and IRAK-2 expression in interleukin-1β (IL-1β) and amyloid-β-42 (Aβ42) peptide-stressed HAG cells. NF-κB-mediated transcriptional control of human IRAK-2 was localized to between −119 and +12 bp of the immediate IRAK-2 promoter. The NF-κB inhibitors curcumin, pyrrolidine dithiocarbamate or CAY10512 abrogated both IRAK-2 and miRNA-146a expression, whereas IRAK-1 was up-regulated. Incubation of a protected antisense miRNA-146a was found to inhibit miRNA-146a and restore IRAK-1, whereas IRAK-2 remained unaffected. These data suggest a significantly independent regulation of IRAK-1 and IRAK-2 in AD and in IL-1β+Aβ42 peptide-stressed HAG cells and that an inducible, NF-κB-sensitive, miRNA-146a-mediated down-regulation of IRAK-1 coupled to an NF-κB-induced up-regulation of IRAK-2 expression drives an extensively sustained inflammatory response. The interactive signaling of NF-κB and miRNA-146a further illustrate interplay between inducible transcription factors and pro-inflammatory miRNAs that regulate brain IRAK expression. The combinatorial use of NF-κB inhibitors with miRNA-146a or antisense miRNA-146a may have potential as a bi-pronged therapeutic strategy directed against IRAK-2-driven pathogenic signaling.

Increased expression of miRNA-146a in Alzheimer's disease transgenic mouse models

A mouse and human brain-enriched micro-RNA-146a (miRNA-146a) is known to be important in modulating the innate immune response and inflammatory signaling in certain immunological and brain cell types. In this study we examined miRNA-146a levels in early-, moderate- and late-stage Alzheimers disease (AD) neocortex and hippocampus, in several human primary brain and retinal cell lines, and in 5 different transgenic mouse models of AD including Tg2576, TgCRND8, PSAPP, 3xTg-AD and 5xFAD. Inducible expression of miRNA-146a was found to be significantly up-regulated in a primary co-culture of human neuronal-glial (HNG) cells stressed using interleukin1-beta (IL-1β), and this up-regulation was quenched using specific NF-кB inhibitors including curcumin. Expression of miRNA-146a correlated with senile plaque density and synaptic pathology in Tg2576 and in 5xFAD transgenic mouse models used in the study of this common neurodegenerative disorder.

Alzheimer's disease and the microbiome

The recognition of the human microbiome (HM) as a substantial contributor to nutrition, health and disease is a relatively recent one, and currently, peerreviewed studies linking alterations in microbiota to the etiopathology of human disease are few. Emerging studies indicate that the HM may contribute to the regulation of multiple neuro-chemical and neuro-metabolic pathways through a complex series of highly interactive and symbiotic host-microbiome signaling systems that mechanistically interconnect the gastrointestinal (GI) tract, skin, liver, and other organs with the central nervous system (CNS). For example, the human GI tract, containing 95% of the HM, harbors a genetically diverse microbial population that plays major roles in nutrition, digestion, neurotrophism, inflammation, growth, immunity and protection against foreign pathogens (Forsythe et al., 2012; Collins et al., 2013; Douglas-Escobar et al., 2013; see below). It has been estimated that about 100 trillion bacteria from up to 1000 distinct bacterial species co-inhabit the human GI tract, albeit in different stoichiometries amongst individuals, and the varying combinations and strains of bacterial species amongst human populations might contribute, in part, to “humanbiochemical” or “genetic-individuality” and resistance to disease (Aziz et al., 2013; Lukiw, 2013). Interestingly, HM participation in human physiology may also help explain the genome-complexity conundrum—for example why the 26,600 protein-encoding transcripts in Homo sapiens are far fewer in number, than for example, the rice genome (Oryza sativa; which has about 46,000 functional genes). One thousand different strains of bacteria might be expected to contribute up to 4 × 106 potential mRNAs to the human transcriptome, thus making the human host-plus-microbiome genetic complexity closer to 4,026,600 mRNA transcripts, and a clear “winner” of human genetic complexity over that of rice and other species (Venter et al., 2001; Foster and McVey Neufeld, 2013; Lukiw, 2013). The very recent observation of microbiomederived small non-coding RNA (sncRNA) and micro RNA (miRNA) translocation and signaling across endothelial barriers, between cells and tissues, and even perhaps between individual species indicates that human neurobiology may be significantly impacted by the actions of HMmediated sncRNA or miRNA trafficking, and the integration of a cell, tissue or an entire organism into its local environment (Zhao et al., 2006; Alexandrov et al., 2012; Sarkies and Miska, 2013; Reijerkerk et al., 2013; unpublished). This opinion paper encompasses what we know concerning the contribution of the HM to neurological disease, with specific emphasis on Alzheimer’s disease (AD) wherever possible. Firstly, the microbiome of the human GI tract is the largest reservoir of microbes in the body, containing about 1014 microorganisms; over 99% of microbiota in the gut are anaerobic bacteria, with fungi, protozoa, archaebacteria and other microorganismsmaking up the remainder. There is currently an expanding interest in the ability of intestinal bacteria to influence neuro-immune functions well beyond the GI tract. Since mitochondria are believed to originate from bacteria via endosymbiotic relationships that formed very early in the evolutionary history of eukaryotes, cross-reactivity of mitochondria and immunological responses to intestinal bacterial constituents could have deleterious effects on mitochondrial function through some form of molecular mimicry; this is partially evidenced by the inflammatory basal ganglia disorder Sydenham’s chorea, rheumatic fever and the link to the facultative anaerobe Streptococcus (Carrasco-Pozo et al., 2012; Douglas-Escobar et al., 2013; Hayashi, 2013; Hornig, 2013 see below). Established pathways of GI-CNS communication currently include the autonomic nervous system (ANS), the enteric nervous system (ENS), the neuroendocrine system, and the immune system (Camfield et al., 2011; Heijtz et al., 2011; Forsythe et al., 2012; Aziz et al., 2013; Collins et al., 2013; Foster and McVey Neufeld, 2013; Schwartz and Boles, 2013). Stress further influences the composition of the HM, and reciprocal communication between the CNS and the HM also influences stress reactivity (Forsythe et al., 2012; Foster and McVey Neufeld, 2013). Surprisingly, neuronal signaling pathways along the bidirectional GI-CNS axis remain poorly understood despite their important roles: (i) in coordinating metabolicand nutritivefunctions, and (ii) in their functional disruption in chronic diseases such as metabolic syndrome, diabetes, obesity, anxiety, autoimmune-disease and stressinduced neuropsychiatric disease (Lukiw and Bazan, 2006; Bravo et al., 2012; Foster and McVey Neufeld, 2013; Hornig, 2013; Udit and Gautron, 2013). Studies of the ENS in “germ-free” mice, i.e., those

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.

Spreading of Alzheimer's disease inflammatory signaling through soluble micro-RNA.

&NA; Alzheimers disease is a progressive, neurodegenerative disorder that develops within the limbic system, spreading radially into anatomically linked brain association areas as the disease progresses. Analysis of temporal‐lobe association of neocortex‐derived extracellular fluid and cerebrospinal fluid from Alzheimers disease patients shows an abundant presence of micro‐RNA (miRNA), including the proinflammatory miRNA‐146a and miRNA‐155. Using a novel and highly sensitive LED‐Northern dot‐blot focusing technique, we detected the secretion of potentially pathogenic amounts of miRNA‐146a and miRNA‐155 from stressed human primary neural cells. A conditioned medium containing miRNA‐146a and miRNA‐155 was found to induce Alzheimer‐type gene expression changes in control brain cells. These included downregulation in the expression of an important repressor of the innate immune response, complement factor H (CFH). These effects were neutralized using anti‐miRNA strategies. Anti‐miRNA‐based therapeutics may provide a novel and efficacious treatment to stem the miRNA‐mediated spreading of inflammatory signaling involved in Alzheimers disease.

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.

Up-regulation of NF-kB-sensitive miRNA-125b and miRNA-146a in metal sulfate-stressed human astroglial (HAG) primary cell cultures

Micro RNAs (miRNAs) constitute a unique class of small, non-coding ribonucleic acids (RNAs) that regulate gene expression at the post-transcriptional level. The presence of two inducible miRNAs, miRNA-125b and miRNA-146a, involved in respectively, astroglial cell proliferation and in the innate immune and inflammatory response, is significantly up-regulated in human neurological disorders including Alzheimers disease (AD). In this study we analyzed abundances miRNA-125b and miRNA-146a in magnesium-, iron-, gallium, and aluminum-sulfate-stressed human-astroglial (HAG) cells, a structural and immune-responsive brain cell type. The combination of iron- plus aluminum-sulfate was found to be significantly synergistic in up-regulating reactive oxygen species (ROS) abundance, NF-кB-DNA binding and miRNA-125b and miRNA-146a expression. Treatment of metal-sulfate stressed HAG cells with the antioxidant phenyl butyl nitrone (PBN) or the NF-кB inhibitors curcumin, the metal chelator-anti-oxidant pyrollidine dithiocarbamate (PDTC), or the resveratrol analog CAY10512, abrogated both NF-кB signaling and induction of these miRNAs. Our observations further illustrate the potential of physiologically relevant amounts of aluminum and iron sulfates to synergistically up-regulate specific miRNAs known to contribute to AD-relevant pathogenetic mechanisms, and suggest that antioxidants or NF-кB inhibitors may be useful to quench metal-sulfate triggered genotoxicity.

Regulation of TREM2 expression by an NF-кB-sensitive miRNA-34a.

Genetic deficits and loss of function for the triggering receptor expressed in myeloid cells 2 (TREM2; encoded at chr6p21.1), a transmembrane spanning stimulatory receptor of the immunoglobulin/lectin-like gene superfamily, have been associated with deficiencies in phagocytosis and the innate immune system in Alzheimer’s disease. In this study, we provide evidence that TREM2 is downregulated in samples of sporadic Alzheimer hippocampal CA1 compared with age-matched controls. A nuclear factor-кB (NF-кB)-sensitive miRNA-34a (encoded at chr1p36.22), upregulated in Alzheimer’s disease, was found to target the 299 nucleotide human TREM2 mRNA 3′-untranslated region (3′-UTR) and downregulate the expression of a TREM2-3′-UTR reporter vector. A stabilized anti-miRNA-34a (AM-34a) quenched this pathogenic response. The results suggest that an epigenetic mechanism involving an NF-кB-mediated, miRNA-34a-regulated downregulation of TREM2 expression may shape innate immune and phagocytic responses that contribute to inflammatory neurodegeneration.

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.

Aluminum and its potential contribution to Alzheimer's disease (AD)

Alzheimers disease (AD) is perhaps the principal example of cognitive failure in humans, and currently over 5.5 million Americans suffer from this incapacitating and progressive disorder of thought, reasoning and memory. Our laboratory has been evaluating the potential contribution of environmentally bioavailable neurotoxic metals to the onset, development and progression of AD for about 30 years (Lukiw et al., 1987). Largely because of its known multiple and potent neurotoxic effects, much of our research has focused on the potential contribution of aluminum to the AD process: (i) because of aluminums remarkable abundance and bioavailability in the biosphere—in fact it is the most abundant naturally occurring neurotoxic element to which we are exposed; (ii) because of aluminums remarkable cellular toxicity and genotoxicity at low nanomolar concentrations toward brain genetic processes, and (iii) because of aluminums highly structured, specific and unique interactions with the phosphate-rich nucleic acids associated with the expression of genetic information in the human brain (Lukiw et al., 1989; Bryant et al., 2004; Alexandrov et al., 2005, 2013; Lukiw and Pogue, 2007; Pogue et al., 2009, 2012; Lukiw, 2010; Percy et al., 2011; Bhattacharjee et al., 2013; De Sole et al., 2013). Aluminums contribution to AD is based upon at least seven independently derived observations: (i) that at physiologically realistic concentrations, aluminum strongly promotes amyloid aggregation and accumulation, a key feature of AD neuropathology (Exley, 2005; Rodella et al., 2008; Walton and Wang, 2009; Yumoto et al., 2009); (ii) that both in vitro and in vivo aluminum promotes inflammatory signaling via the pro-inflammatory transcription factor NF-kB, another prominent feature characteristic of AD brain (Bondy, 2013; Walton, 2013); (iii) that out of the many thousands of brain gene messenger RNA (mRNAs) and micro RNAs (miRNAs), the family of mRNAs and miRNAs induced by aluminum are also strikingly similar to those found to be increased in AD; (iv) that in transgenic animal models of AD dietary aluminum enhances the development of pathological markers such as lipid peroxidation, oxidative stress, apoptosis, and gene expression deficits (Pratico et al., 2002; Bharathi et al., 2008; Zhang et al., 2012); (v) that many of the observed deficits in AD such as chromatin compaction, impaired energy utilization, impaired signaling involving chemical messengers such as adenine triphosphate (ATP) are recapitulated in aluminum-treated cellular or animal models of AD (Alexandrov et al., 2005; Lukiw and Pogue, 2007; Pogue et al., 2012; Bhattacharjee et al., 2013); (vi) that a very significant number of studies link the amount of aluminum in drinking water to the incidence of AD [worldwide, aluminum is added to drinking water as hydrated aluminum potassium sulfate KAl(SO4)2·12H2O, or alum, as a clarification and “finishing” agent (Flaten, 2001; Frisardi et al., 2010; Walton, 2013)], and (vii) perhaps most importantly, that of all pharmaceutical treatment approaches directed against AD to date, chelation using the anti-oxidant and trivalent iron/aluminum chelator desferrioxamine has been shown to be one of the most effective therapeutic strategies yet devised (Crapper McLachlan et al., 1991; Percy et al., 2011). Abundant research indicates that aluminum is a particularly reactive metal toward multiple aspects of human neurobiology and the altered genetics that are associated with the development and propagation of sporadic AD (Lukiw et al., 1989; Lukiw, 2010; Bhattacharjee et al., 2013; Bondy, 2013; Shaw and Tomljenovic, 2013; Walton, 2013). Thirty years of research since the potent effects of aluminum on the genetic apparatus in AD were first described, the most recent evidence suggests a strong linkage between aluminum sulfates and induction of NF-kB-sensitive pro-inflammatory miRNAs (Lukiw et al., 1987; Alexandrov et al., 2013; Zhao et al., 2013). Aluminum has been previously shown to significantly induce the transcription factor NF-kB (Pogue et al., 2009; Bondy, 2013), and up-regulation of NF-kB drives synthesis of NF-kB-sensitive miRNAs which in turn down regulate the expression of many AD-relevant genes, including complement factor H (CFH) and neurotropic signaling in human brain cells (Pogue et al., 2009; Zhao et al., 2013). We would like here to briefly include some recent genetic data on aluminum and its effects on miRNA abundance in a highly relevant transgenic animal model for AD that shows strong parallels to miRNA profiles which are found in AD brain (Figure ​(Figure1).1). There are currently over 90 transgenic mouse models of AD (http://www.alzforum.org/research-models/). A commonly used Tg2576 mouse model overexpresses a mutant form of beta amyloid precursor protein (β APP), APPK670/671L, linked to early-onset familial AD, and develops amyloid plaques and progressive cognitive deficits as the mice age. Tg2576 mice exposed to dietary aluminum have been shown to develop oxidative stress and robust amyloidogenesis, key features of AD neuropathology (Pratico et al., 2002). Animals provided a 2 mg/kg aluminum-supplemented diet were analyzed for miRNA speciation and complexity in their brains using GeneChip and miRNA array technologies; intriguingly, the same quintet of up-regulated pro-inflammatory miRNAs (miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155) as found in (i) AD and (ii) in aluminum-treated human brain cells in primary culture were also found to be amongst the most up-regulated in these aluminum-supplemented Tg2576 mice (Alexandrov et al., 2005; Lukiw and Pogue, 2007; Pogue et al., 2012; Bhattacharjee et al., 2013; Hill et al., 2014; Figure ​Figure1;1; unpublished observations). Up-regulated miRNAs are known to target susceptible mRNAs and down-regulate the expression of many AD-relevant brain genes as is widely observed in AD brain tissues (Colangelo et al., 2002; Guo et al., 2010; Ginsberg et al., 2012). Interestingly, these findings suggest some common miRNA-induced mechanism between two important in vitro and in vivo models for AD with AD itself. Indeed, the abundance of specific miRNAs are highly selective, and potential indicators and predictors of human health and disease, including progressive neurological disorders such as AD (Alexandrov et al., 2005, 2013; Lukiw and Pogue, 2007; Pogue et al., 2009; Maciotta et al., 2013; Zhao et al., 2013). Figure 1 Array-based cluster analysis of miRNA abundance in aluminum-fed Tg2576 mice vs. controls. In these experiments the brain (cortex) of 3 month-old Tg2576 mice fed aluminum-enriched diets were analyzed for miRNA speciation compared to age-matched controls ... Lastly, more research into the potential contribution of aluminum to the AD process is clearly warranted. There are currently no treatments for AD that effectively prevent or cure ADs insidious onset or propagation. We think it important to emphasize that the most effective clinical treatment yet devised for moderate- to late-stage AD patients was the implementation of the first generation anti-oxidant and trivalent iron/aluminum chelator desferrioxamine to attempt to remove aluminum from the brains of AD patients (Crapper McLachlan et al., 1991; Percy et al., 2011). Second generation aluminum chelators such as Feralex-G, either alone or in combination with other chelators, has shown higher specificity, increased selectivity and higher efficacy in aluminum sequestration and chelation in preliminary in vitro studies (Kruck et al., 2004, 2008; Percy et al., 2011; unpublished observations). Certainly, laboratory experimentation in cultured primary human brain cells, in transgenic AD animal models, and clinical studies employing next-generation aluminum chelators, perhaps in combination with other drug strategies, are one of the research areas needing more focused attention—to more effectively address the exact role and mechanism of aluminum neurotoxicity in this rapidly expanding healthcare concern.