Walter J. Lukiw

Novel docosanoids Inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFκB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-β-induced NFκB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion.

A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease

Deficiency in docosahexaenoic acid (DHA), a brain-essential omega-3 fatty acid, is associated with cognitive decline. Here we report that, in cytokine-stressed human neural cells, DHA attenuates amyloid-beta (Abeta) secretion, an effect accompanied by the formation of NPD1, a novel, DHA-derived 10,17S-docosatriene. DHA and NPD1 were reduced in Alzheimer disease (AD) hippocampal cornu ammonis region 1, but not in the thalamus or occipital lobes from the same brains. The expression of key enzymes in NPD1 biosynthesis, cytosolic phospholipase A2 and 15-lipoxygenase, was altered in AD hippocampus. NPD1 repressed Abeta42-triggered activation of proinflammatory genes while upregulating the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1). Soluble amyloid precursor protein-alpha stimulated NPD1 biosynthesis from DHA. These results indicate that NPD1 promotes brain cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Abeta42-induced neurotoxicity.

An NF-κB-sensitive Micro RNA-146a-mediated Inflammatory Circuit in Alzheimer Disease and in Stressed Human Brain Cells

Human brains retain discrete populations of micro RNA (miRNA) species that support homeostatic brain gene expression functions; however, specific miRNA abundance is significantly altered in neurological disorders such as Alzheimer disease (AD) when compared with age-matched controls. Here we provide evidence in AD brains of a specific up-regulation of an NF-κB-sensitive miRNA-146a highly complementary to the 3′-untranslated region of complement factor H (CFH), an important repressor of the inflammatory response of the brain. Up-regulation of miRNA-146a coupled to down-regulation of CFH was observed in AD brain and in interleukin-1β, Aβ42, and/or oxidatively stressed human neural (HN) cells in primary culture. Transfection of HN cells using an NF-κB-containing pre-miRNA-146a promoter-luciferase reporter construct in stressed HN cells showed significant up-regulation of luciferase activity that paralleled decreases in CFH gene expression. Treatment of stressed HN cells with the NF-κB inhibitor pyrollidine dithiocarbamate or the resveratrol analog CAY10512 abrogated this response. Incubation of an antisense oligonucleotide to miRNA-146a (anti-miRNA-146a; AM-146a) was found to restore CFH expression levels. These data indicate that NF-κB-sensitive miRNA-146a-mediated modulation of CFH gene expression may in part regulate an inflammatory response in AD brain and in stressed HN cell models of AD and illustrate the potential for anti-miRNAs as an effective therapeutic strategy against pathogenic inflammatory signaling.

Docosahexaenoic Acid and the Aging Brain

The dietary essential PUFA docosahexaenoic acid [DHA; 22:6(n-3)] is a critical contributor to cell structure and function in the nervous system, and deficits in DHA abundance are associated with cognitive decline during aging and in neurodegenerative disease. Recent studies underscore the importance of DHA-derived neuroprotectin D1 (NPD1) in the homeostatic regulation of brain cell survival and repair involving neurotrophic, antiapoptotic and antiinflammatory signaling. Emerging evidence suggests that NPD1 synthesis is activated by growth factors and neurotrophins. Evolving research indicates that NPD1 has important determinant and regulatory interactions with the molecular-genetic mechanisms affecting beta-amyloid precursor protein (betaAPP) and amyloid beta (Abeta) peptide neurobiology. Deficits in DHA or its peroxidation appear to contribute to inflammatory signaling, apoptosis, and neuronal dysfunction in Alzheimer disease (AD), a common and progressive age-related neurological disorder unique to structures and processes of the human brain. This article briefly reviews our current understanding of the interactions of DHA and NPD1 on betaAPP processing and Abeta peptide signaling and how this contributes to oxidative and pathogenic processes characteristic of aging and AD pathology.

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.

Docosahexaenoic Acid-Derived Neuroprotectin D1 Induces Neuronal Survival via Secretase- and PPARγ-Mediated Mechanisms in Alzheimer's Disease Models

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimers disease (AD) brain. Here we further characterize the mechanism of NPD1s neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)sw (Swedish double mutation APP695sw, K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations.

Circular RNA (circRNA) in Alzheimer's disease (AD)

Circular RNAs (circRNAs) are a naturally occurring family of noncoding RNAs (ncRNAs) highly represented in the eukaryotic transcriptome. Recently characterized, traditional methods of RNA detection and analysis requiring a free 5′ or 3′ ribonucleotide terminus may have significantly underestimated circRNA abundance and significance in eukaryotic cells (Salzman et al., 2012; Wilusz and Sharp, 2013; unpublished observations). Intrinsically resistant to exonucleolytic RNA decay, circRNAs appear to be enriched in mammalian brain tissues (Hansen et al., 2013; Memczak et al., 2013). Interestingly, specific ncRNAs such as the evolutionary ancient microRNA-7 (miRNA-7; chr 9q21.32; an important post-transcriptional regulator of human brain gene expression), are not only highly abundant in human brain, but are also associated with a circRNA for miRNA-7 (ciRS-7), in the same tissues; ciRS-7 contains multiple, tandem anti-miRNA-7 sequences (Burmistrova et al., 2007; Hansen et al., 2013; Lukiw et al., 2013). ciRS-7 thereby acts as a kind of endogenous, competing, anti-complementary miRNA “sponge” to adsorb, and hence quench, normal miRNA-7 functions. Using Northern blot hybridization techniques and the circularity-sensitive circRNA probe RNaseR we here provide initial evidence of a mis-regulated miRNA-7-circRNA system in the sporadic Alzheimers disease (AD) hippocampal CA1 region (Figure ​(Figure1).1). Deficits in ciRS-7, and ciRS-7 “sponging activities” might be expected to increase ambient miRNA-7 levels in AD-affected brain cells, as is observed, to ultimately contribute to the down-regulation of selective miRNA-7-sensitive messenger RNA (mRNA) targets (Cogswell et al., 2008; unpublished observations). The presence of up-regulated miRNA-7, due to a deficiency in ciRS-7 “sponging” effects, has high probability to down-regulate AD-relevant targets, such as, for example, the ubiquitin protein ligase A (UBE2A; miRNA-7-UBE2A mRNA energy of association, EA = −22.86 kcal/mol). UBE2A, an autophagic, phagocytic protein essential in the clearance of amyloid peptides in AD and other progressive inflammatory degenerations of the human CNS, is depleted in AD brain (Bingol and Sheng, 2011; Lonskaya et al., 2013). Such miRNA-mRNA regulatory systems mediated by a family of cell- and/or tissue-enriched circRNAs may represent another important layer of epigenetic control over gene expression in health and disease. Indeed, technological advancement and recent discoveries in the field of ncRNAs continue to challenge our basic doctrines of nucleic acid biochemistry and evolutionary biology. Deficits in other circRNA-mediated “miRNA sponging systems” and ambient up-regulation of specific inducible miRNAs may help explain the widely observed, generalized and progressive down-regulation of gene expression that is characteristic of the sporadic AD brain (Loring et al., 2001; Colangelo et al., 2002; Ginsberg et al., 2012; Lukiw, 2013). Figure 1 (A) Detection of circRNA for miRNA-7 (ciRS-7) in sporadic Alzheimers disease (AD) and age-matched control hippocampal CA1 [control (C) N = 4; AD (A) N = 6]; the single upper ciRS-7 (~1400 nt) band contains ~70 selectively conserved miRNA-7 binding sites ...

Gene expression profiling in fetal, aged, and Alzheimer hippocampus: a continuum of stress-related signaling.

While specific components of normal brain aging and Alzheimers disease (AD) appear to be genetically determined, it is not well understood whether AD is due to accelerated aging or if AD represents an independent disease entity superimposed upon senescence. Using gene expression profiling, significant alterations in brain-specific transcription patterns have been observed between AD and age-matched controls. In AD, although a general depression in brain genetic output has been reported, there are robust increases in the expression of potentially neuropathological genes. The data in this study show increases in the RNA abundance patterns for a stress-response, proinflammatory, apoptotic, and angiogenic gene family that occur during the transition from fetal to aged, and again during the transformation from aged to AD brain. Significantly up-regulated RNAs include those encoding stress-induced factors (HSP70), transcriptional repressors (DAXX), pentraxins (SAP), proapoptosis factors (FAS and DAXX), and several inflammatory markers (βAPP, CEX1, NF-IL6, NF-kappaBp100, cyclooxygenase-2, IL-1α and IL-1β precursors and cPLA2). These findings support the hypothesis that there is a continuum of stress-related gene expression as the brain ages and an advancement of inflammatory, apoptotic, and angiogenic gene signaling that correlates with the transition to AD.

Metallothionein III is reduced in Alzheimer's disease.

Metallothionein III (MT-III) is a functionally distinct member of the metallothionein family that displays neuroinhibitory activity and is involved in the repair of neuronal damage. Altered expression levels of MT-III have been observed in Alzheimers disease (AD) which has led to suggestions that it could be a mitigating factor in AD-related neuronal dysfunction. However, conflicting results have been reported on this issue which may be due to methodological differences and/or sampling size. In the current study, we have assessed MT-III expression in a large number of AD cases through the quantification of mRNA as well as by immunohistochemistry and Western blotting using an MT-III specific antibody. The results of this comprehensive study indicate that the mononucleosome DNA encoding MT-III is occluded preventing transcription and that message levels are reduced by approximately 30%. In addition, protein levels were specifically decreased by approximately 55% in temporal cortex. These data support the conclusion that MT-III is significantly downregulated in AD and may contribute to the loss of its protective effects and/or repair functions that lead to an exacerbation of the pathogenic processes.

Cyclooxygenase 2 RNA message abundance, stability, and hypervariability in sporadic Alzheimer neocortex.

Long‐term treatment by nonsteroidal anti‐inflammatory drugs has been shown to decrease the incidence of Alzheimers disease (AD). Both platelet‐activating factor and interleukin‐1β, potent mediators of the inflammatory and immune response, strongly induce transcription of the cyclooxygenase‐2 (COX‐2) gene in brain cells. Using Northern and RT‐PCR analysis, we have determined in 15 control and 10 sporadic AD human neocortical samples (age range, 60–82 yr; postmortem interval [PMI] range, 0.7–16.0 hr) the levels of COX‐2 RNA in relation to the constitutively expressed COX‐1 and β‐actin RNA message levels. Our results indicate that in short PMI brain, COX‐1 and COX‐2 transcripts are relatively low abundance RNA messages, ranging from a mean of 6.8% of the β‐actin signal in controls to 8.5% of the β‐actin signal in AD‐affected brain. A large variation in the signal intensity for COX‐2 RNA was noted in both control and AD; although there was a trend for higher COX‐2 RNA message abundance in AD neocortex to +11.5% of that of controls, it did not reach statistical significance (ANOVA = 0.45). Several human tissues, including heart, skeletal muscle, lung, kidney, and spinal cord, displayed 4.6‐ and 2.8‐kb COX‐2 RNA message isoforms; however, the 4.6‐kb COX‐2 RNA predominated in the hippocampus and association neocortex. COX‐2 RNA message was found to be degraded at similar rates in both control and AD tissues, and a strong positive correlation between the PMI and the intensity of the COX‐2 RNA signal was noted (ANOVA = 0.006). Linear regression analysis indicated that the 4.6‐kb COX‐2 RNA is an unstable short‐lived RNA species with a half‐life of not more than 3.5 hr, a feature characteristic of immediate early gene transcripts. Individual hypervariability in COX‐2 RNA message abundance may reflect various degrees of expression of AD‐related inflammatory processes. J. Neurosci. Res. 50:937–945, 1997. © 1997 Wiley‐Liss, Inc.