Prerna Dua

UPREGULATION OF MICRO RNA-146a (miRNA-146a), A MARKER FOR INFLAMMATORY NEURODEGENERATION, IN SPORADIC CREUTZFELDT-JAKOB DISEASE (sCJD) AND GERSTMANN–STRAUSSLER–SCHEINKER (GSS) SYNDROME

A mouse- and human-brain-abundant, nuclear factor (NF)-кB-regulated, micro RNA-146a (miRNA-146a) is an important modulator of the innate immune response and inflammatory signaling in specific immunological and brain cell types. Levels of miRNA-146a are induced in human brain cells challenged with at least five different species of single- or double-stranded DNA or RNA neurotrophic viruses, suggesting a broad role for miRNA-146a in the brains innate immune response and antiviral immunity. Upregulated miRNA-146a is also observed in pro-inflammatory cytokine-, Aβ42 peptide- and neurotoxic metal-induced, oxidatively stressed human neuronal-glial primary cell cocultures, in murine scrapie and in Alzheimers disease (AD) brain. In AD, miRNA-146a levels are found to progressively increase with disease severity and co-localize to brain regions enriched in inflammatory neuropathology. This study provides evidence of upregulation of miRNA-146a in extremely rare (incidence 1–10 per 100 million) human prion-based neurodegenerative disorders, including sporadic Creutzfeldt–Jakob disease (sCJD) and Gerstmann–Straussler–Scheinker syndrome (GSS). The findings suggest that an upregulated miRNA-146a may be integral to innate immune or inflammatory brain cell responses in prion-mediated infections and to progressive and irreversible neurodegeneration of both the murine and human brain.

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.

Microbial Sources of Amyloid and Relevance to Amyloidogenesis and Alzheimer’s Disease (AD)

Since the inception of the human microbiome project (HMP) by the US National Institutes of Health (NIH) in 2007 there has been a keen resurgence in our recognition of the human microbiome and its contribution to development, immunity, neurophysiology, metabolic and nutritive support to central nervous system (CNS) health and disease. What is not generally appreciated is that (i) the ~1014 microbial cells that comprise the human microbiome outnumber human host cells by approximately one hundred-to-one; (ii) together the microbial genes of the microbiome outnumber human host genes by about one hundred-and-fifty to one; (iii) collectively these microbes constitute the largest ‘diffuse organ system’ in the human body, more metabolically active than the liver; strongly influencing host nutritive-, innate-immune, neuroinflammatory-, neuromodulatory- and neurotransmission-functions; and (iv) that these microbes actively secrete highly complex, immunogenic mixtures of lipopolysaccharide (LPS) and amyloid from their outer membranes into their immediate environment. While secreted LPS and amyloids are generally quite soluble as monomers over time they form into highly insoluble fibrous protein aggregates that are implicated in the progressive degenerative neuropathology of several common, age-related disorders of the human CNS including Alzheimer’s disease (AD). This general commentary-perspective paper will highlight some recent findings on microbial-derived secreted LPS and amyloids and the potential contribution of these neurotoxic and proinflammatory microbial exudates to age-related inflammatory amyloidogenesis and neurodegeneration, with specific reference to AD wherever possible.

microRNA-34a-Mediated Down-Regulation of the Microglial-Enriched Triggering Receptor and Phagocytosis-Sensor TREM2 in Age-Related Macular Degeneration.

The aggregation of Aβ42-peptides and the formation of drusen in age-related macular degeneration (AMD) are due in part to the inability of homeostatic phagocytic mechanisms to clear self-aggregating Aβ42-peptides from the extracellular space. The triggering receptor expressed in myeloid/microglial cells-2 (TREM2), a trans-membrane-spanning, sensor-receptor of the immune-globulin/lectin-like gene superfamily is a critical component of Aβ42-peptide clearance. Here we report a significant deficit in TREM2 in AMD retina and in cytokine- or oxidatively-stressed microglial (MG) cells. RT-PCR, miRNA-array, LED-Northern and Western blot studies indicated up-regulation of a microglial-enriched NF-кB-sensitive miRNA-34a coupled to a down-regulation of TREM2 in the same samples. Bioinformatics/transfection-luciferase reporter assays indicated that miRNA-34a targets the 299 nucleotide TREM2-mRNA-3’UTR, resulting in TREM2 down-regulation. C8B4-microglial cells challenged with Aβ42 were able to phagocytose these peptides, while miRNA-34a down-regulated both TREM2 and the ability of microglial-cells to phagocytose. Treatment of TNFα-stressed MG cells with phenyl-butyl nitrone (PBN), caffeic-acid phenethyl ester (CAPE), the NF-B-inhibitor/resveratrol analog CAY10512 or curcumin abrogated these responses. Incubation of anti-miRNA-34a (AM-34a) normalized miRNA-34a abundance and restored TREM2 back to homeostatic levels. These data support five novel observations: (i) that a ROS- and NF-B-sensitive, miRNA-34a-mediated modulation of TREM2 may in part regulate the phagocytic response; (ii) that gene products encoded on two different chromosomes (miRNA-34a at chr1q36.22 and TREM2 at chr6p21.1) orchestrate a phagocytic-Aβ42-peptide clearance-system; (iii) that this NF-kB-mediated-miRNA-34a-TREM2 mechanism is inducible from outside of the cell; (iv) that when operating normally, this pathway can clear Aβ42 peptide monomers from the extracellular medium; and (v) that anti-NF-kB and/or anti-miRNA (AM)-based therapeutic strategies may be useful against deficits in TREM-2 receptor-based-sensing and -phagocytic signaling that promote pathogenic amyloidogenesis.

Up-Regulation of miRNA-146a in Progressive, Age-Related Inflammatory Neurodegenerative Disorders of the Human CNS

1 Russian Academy of Medical Sciences, Moscow, Russia 2 Department of Health Information Management, Louisiana State University, Ruston, LA, USA 3 Department of Neurology, Louisiana State University Health Science Center, New Orleans, LA, USA 4 LSU Neuroscience Center and Department of Ophthalmology, Louisiana State University Health Science Center, New Orleans, LA, USA *Correspondence: wlukiw@lsuhsc.edu

Regulating amyloidogenesis through the natural triggering receptor expressed in myeloid/microglial cells 2 (TREM2)

Amyloidogenesis, the progressive accumulation of amyloid-beta (Aβ) peptides into insoluble, toxic, senile plaque lesions is one of the major defining features of the Alzheimers disease (AD) brain. Normally, Aβ42 peptides are cleared from the extracellular space by natural phagocytic mechanisms, but when this intrinsic sensing and clearance system is functionally compromised or defective, Aβ42 peptides accumulate. Largely due to their intensely hydrophobic character, under physiological conditions Aβ42 peptides have a strong tendency to self-aggregate into higher order neurotoxic and pro-inflammatory fibrillar aggregates. While the Aβ peptide-clearing mechanisms are highly complex, one particularly important molecular sensor for Aβ42 peptide clearance is the triggering receptor expressed in myeloid/microglial cells 2 (TREM2; encoded at chr6p21.1), a variably glycosylated 230 amino acid transmembrane spanning stimulatory receptor of the immunoglobulin/lectin-like gene superfamily strongly associated with innate-immune, pro-inflammatory, and neurodegenerative signaling in AD. TREM2 is highly and almost exclusively expressed on the outer plasma membrane of microglial cells, the resident phagocytic and scavenging neuroimmune macrophages of the human central nervous system (CNS). As AD progresses, microglia appear to become progressively dysfunctional; TREM2 becomes down-regulated and microglia lose their ability to clear Aβ42 peptides while producing and releasing neurotoxins, reactive oxygen species (ROS), and pro-inflammatory cytokines that further promote Aβ42 production and pathological aggregation (Schmid et al., 2002; Alexandrov et al., 2013; Boutajangout and Wisniewski, 2013; Hickman and El Khoury, 2013). Recently, much interest in the molecular biology, genetics, and epigenetics of TREM2 expression, and its potential for sensing and scavenging Aβ42 peptides in AD and other progressive neurodegenerative diseases has arisen. TREM2s critical importance is underscored by seven recent observations: (1) that relatively rare mutations of TREM2 (or of its coupling protein, DAP12, also known as TYROBP; see Figure ​Figure1)1) are currently associated with the progressive, pre-senile dementing illnesses Nasu-Hakola syndrome, polycystic lipomembranous osteodysplasia with sclerosing leucoencephalopathy (POSL), sporadic amyotrophic lateral sclerosis (ALS), and AD (Nimmerjahn et al., 2005; Neumann and Takahashi, 2007; Guerreiro and Hardy, 2013; Zhao and Lukiw, 2013; Zhao et al., 2013; Cady et al., 2014); (2) that down-regulation in the phagocytic ability of microglia to degrade Aβ42 peptides in AD, and down-regulation in TREM2 expression, has been reported in sporadic AD brain tissues (Hickman and El Khoury, 2013; Zhao et al., 2013); (3) that TREM2 knockdown has been shown to exacerbate age-related neuro-inflammation and enhance cognitive deficiency in senescence accelerated mouse prone 8 (SAMP8) mice (Jiang et al., 2014); (4) that microglial TREM2 gene expression in cell culture, both at the level of mRNA and protein, have been shown to be remarkably sensitive to external cytokine stressors such as tumor necrosis factor alpha (TNFα; a pro-inflammatory adipokine known to be up-regulated in AD brain; Zhao et al., 2013; unpublished observations); (5) that AD-relevant pro-inflammatory neurotoxins such as bacterial lipopolysaccharide (LPS) and environmentally abundant toxic metals such as aluminum strongly down-regulate TREM2 and the ability of microglial cells to phagocytose extracellular debris (Hickman and El Khoury, 2013; Zhao et al., 2013; unpublished observations); (6) that down-regulation in the expression of TREM2 appears to be regulated in part by the up-regulation of the microglial-enriched, NF-kB-sensitive microRNA-34a (miRNA-34a), and perhaps other NF-kB-sensitive miRNAs, and (7) that both anti-NF-kB and anti-microRNA (AM-RNA) strategies have been shown to be useful in the restoration of homeostatic TREM2 gene expression levels and the neutralization of pro-inflammatory signaling and amyloidogenesis, at least in vitro (Hill et al., 2009; Pogue et al., 2009, 2010; Alexandrov et al., 2012, 2013; Zhao et al., 2013; unpublished observations). From what we know so far it is tempting to speculate that (1) loss-of function of TREM2 due to genetic mutations in familial AD may have the same end effects on phagocytosis as down-regulation of a fully functional TREM2 in sporadic AD; and that (2) modest TREM2 over-expression might be useful in enhancing the scavenging and removal of cellular debris in the CNS, including neurotoxic and self-aggregating Aβ42 peptides. Importantly, TREM2 signaling has been recently shown to be selectively inducible and manipulated from outside of the cell, suggesting that the modulation of TREM2 expression may be effectively regulated using highly specific targeting via drug-based pharmacological strategies exogenously supplied (Alexandrov et al., 2013; Lukiw, 2013; Zhao et al., 2013). Figure 1 The triggering receptor expressed in myeloid/microglial cells type 2 (TREM2) is a 230 amino acid, ~25.4 kDa integral trans-membrane glycoprotein sensor spanning the lipid bilayer of CNS microglial cells. Very recently, TREM2 has been shown to act as a ... AD represents a highly complex, insidious, progressive, multi-factorial brain dysfunction whose incidence is reaching epidemic proportions. Despite the billions of dollars already spent on AD research, including multiple Aβ immunization and immunotherapy strategies, there is still no adequate treatment or cure for AD, and the development and implementation of novel, more effective treatment strategies are critical. Recruitment and harvesting of the TREM2 mechanism as a potent, endogenous Aβ42-peptide scavenging activity may represent a singularly attractive new direction for the clinical management of AD. Indeed, as a natural sensor and scavenger of noxious cellular debris TREM-2 stimulation may turn out (1) to be remarkably neuroprotective against both amyloidogenesis and age-related neuro-inflammation; while (2) significantly reducing the progressive cognitive impairment associated with amyloidogenesis and inflammatory neurodegeneration in the CNS (Hickman and El Khoury, 2013; Zhao et al., 2013; Cady et al., 2014; Jiang et al., 2014). Put another way, a decline in TREM2s contribution to the innate immune response, in part driving amyloid-clearance deficits and progressive degeneration characteristic of the AD process, suggest novel therapeutic targets and treatment strategies directed at maintaining natural and homeostatic TREM2 functions. This may be accomplished not only through the direct stimulation of TREM2 itself but also through the poorly understood downstream TREM2-linked TYROBP (DAP12) signaling pathways responsible for; (1) the actual phagocytosis of extracellular molecules; (2) the production of damaging quantities of ROS; (3) the induction of pro-inflammatory signals via NF-kB; and (4) the maintenance of homeostatic microglial function that together may diminish amyloidogenesis and the intercellular propagation of pathogenic signaling in the AD affected brain (Boutajangout and Wisniewski, 2013; Guerreiro and Hardy, 2013; Zhao and Lukiw, 2013; Jiang et al., 2014; Figure ​Figure11).

Beta-Amyloid Precursor Protein (βAPP) Processing in Alzheimer's Disease (AD) and Age-Related Macular Degeneration (AMD).

Amyloid is a generic term for insoluble, often intensely hydrophobic, fibrous protein aggregates that arise from inappropriately folded versions of naturally-occurring polypeptides. The abnormal generation and accumulation of amyloid, often referred to as amyloidogenesis, has been associated with the immune and pro-inflammatory pathology of several progressive age-related diseases of the human central nervous system (CNS) including Alzheimer’s disease (AD) and age-related macular degeneration (AMD). This ‘research perspective’ paper reviews some of the research history, biophysics, molecular-genetics and environmental factors concerning the contribution of amyloid beta (Aβ) peptides, derived from beta-amyloid precursor protein (βAPP), to AD and AMD that suggests an extensive similarity in immune and inflammatory degenerative mechanisms between these two CNS diseases.

microRNA-Based Biomarkers and the Diagnosis of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized as a complex, age-related neurological disorder of the human central nervous system (CNS) that involves the progressive mis-regulation of multiple biological pathways at multiple molecular, genetic, epigenetic, neurophysiological, cognitive, and behavioral levels. It has been about 8 years since the first reports of altered microRNA (miRNA) abundance and speciation: (i) in anatomical regions of the brain targeted by the AD process after post-mortem examination, (ii) in blood serum, and (iii) in cerebrospinal fluid (CSF) (1–3). Since then an in depth overview of the peer-reviewed literature has provided no general consensus of what miRNAs are up-or-down regulated in any tissue or biofluid compartment in thousands of AD patients. In this brief “Opinion” paper on “Biomarkers of Alzheimer’s disease: the present and the future,” we will highlight the extremely heterogeneous nature of miRNA expression in AD, based on very recent advances in the analysis of miRNA populations in various biofluid compartments compared to normally aging, neurologically normal controls. This work is based against a background of our laboratory’s 24 years of research experience into the structure and function of small, non-coding RNAs in the aging human CNS in health and in age-related neurological disease (4).

An evaluation of progressive amyloidogenic and pro-inflammatory change in the primary visual cortex and retina in Alzheimer's disease (AD)

James M. Hill , Prerna Dua, Christian Clement and Walter J. Lukiw* 1 Louisiana State University Neuroscience Center and Departments of Ophthalmology and Pharmacology, Louisiana State University Health Science Center, New Orleans, LA, USA 2 Department of Health Information Management, Louisiana State University, Ruston, LA, USA 3 Department of Natural Sciences, Infectious Diseases, Experimental Therapeutics and Human Toxicology Lab, Southern University at New Orleans, New Orleans, LA, USA 4 Department of Neurology, Louisiana State University Health Science Center, New Orleans, LA, USA *Correspondence: wlukiw@lsuhsc.edu

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