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.


Introduction
Currently affecting about 150 million individuals worldwide, age-related macular degeneration (AMD) is a common, neurodegenerative disorder of the human retina characterized clinically by the progressive erosion of central vision [1,2]. AMD is further subdivided into a "wet" form, involving choroidal neovascularization, and the much more common "dry" form of AMD, characterized by the presence of yellow lipoprotein-rich deposits, called drusen, in the macula, the central portion of the retina. The drusen of AMD typically develop with aging and contain a beta-amyloid precursor protein (βAPP)-derived 42 amino acid amyloid beta peptide (Aβ42) as a major component [3][4][5]. The molecular-genetic mechanisms regulating Aβ42 peptide accumulation and clearance are not completely understood, but appear to involve a receptormediated sensing of Aβ42 peptide monomers and other toxic molecules in the extracellular space as an initial step in phagocytosis and homeostatic clearance. One prominent sensorreceptor for Aβ42-peptide clearance in the CNS is the triggering receptor expressed in myeloid/microglial cells-2 (TREM2; chr6p21.1), a~230 amino acid, single pass type 1 transmembrane sensor-receptor protein enriched in the plasma membrane of microglial (MG) cells [6][7][8][9][10][11]. Mutations and loss-of-function for TREM2 have been associated with deficiencies in phagocytosis, the innate-immune system, axonal and synaptic abnormalities, amyloidogenesis and progressive dementia in progressive neurological diseases of the human CNS including polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL; also known as Nasu-Hakola disease [6][7][8][9][10][11] as well as more recently in sporadic amyotrophic lateral sclerosis (ALS) [11] and in Alzheimer's disease (AD) [6][7][8][9][10][11][12][13][14][15].
Micro RNAs (miRNAs) are~22 nucleotide, non-coding RNA single stranded (ssRNA) molecules that represent a family of heterogeneous, evolutionarily conserved, regulatory RNAs that recognize the 3' un-translated region (3'UTR) of specific messenger RNA (mRNA) targets [16,17]. In doing so miRNAs down-regulate the post-transcriptional stability or translational efficiency of their target mRNAs, thus functioning as natural negative regulators of gene expression [16][17][18][19]. Of the~2650 human miRNAs so far identified: (i) only a specific subset of miRNAs are highly expressed in the CNS; (ii) many of these are critical to the regulation of normal brain and retinal cell function in health and aging; and (iii) many of these miRNAs appear to be inducible by age-related pathological and environmental factors [17][18][19][20][21]. Like neurons and astroglia, MG cells express a select family of miRNAs that support homeostatic retinal gene expression functions and specific miRNA abundances and are significantly altered in AMD-affected retina when compared to age-matched controls [20][21][22][23][24]. As few miRNAs have been functionally linked to specific retinal pathways involving phagocytosis, these studies were undertaken to further understand the involvement of specific, retinal-enriched, inducible miRNAs in the molecular-genetic mechanism that drives amyloidogenesis, TREM2 down-regulation, drusen formation and AMD-type change. Here we provide evidence that in human AMD and stressed MG cells there occurs a selective up-regulation of an inducible, NF-kB-regulated miRNA-34a coupled to a significant down-regulation of TREM2 expression. Bioinformatics and transfection experiments indicate that miRNA-34a targets the 299 nucleoide (nt) TREM2 mRNA 3'UTR and significantly down-regulates TREM2 expression in MG cells. Transfection of C8B4 MG cells using TREM2 3'UTR luciferase reporter constructs showed specific and significant interaction with miRNA-34a but not with other scrambled (SC) and/or negative control (NC) miRNA-34a sequences. Further, up-regulation of the pro-inflammatory miRNA-34a and TREM2 down-regulation was also observed in reactive oxygen species (ROS)-, IL-1β-and TNFα-stressed MG cell cultures, an effect that could be significantly reversed using anti-ROS, anti-NF-kB and/or anti-miRNA-34a therapeutic strategies. These studies provide 5 new observations: (i) that gene products encoded on two different chromosomes (miRNA-34a at chr1q36.22 and TREM2 at chr6p21.1) coordinate a phagocytosis-mediated Aβ42-peptide clearance system in stressed-MG cells and in sporadic AMD retina; (ii) that increased expression of a ROS-induced, inflammatory cytokine and NF-kB-sensitive MGenriched miRNA-34a down-regulates a specific mRNA target encoding TREM2, a membranespanning glycoprotein known to contribute to the clearance of pro-inflammatory Aβ42 peptides; (iii) that a miRNA-34a-regulated TREM2 circuit can clear low molecular weight Aβ42 peptides from the extracellular medium; (iv) that this miRNA-34a-TREM2 clearance system is inducible from outside of the cell; and (v) that anti-ROS, anti-miRNA or anti-NF-kB pharmacological strategies may be useful in the clinical management of deficits in phagocytic signaling that drive pathogenic amyloidogenesis. These findings suggest that an epigenetic mechanism involving an NF-kB-mediated, miRNA-34a-regulated down-regulation of TREM2 expression may shape innate-immune and phagocytic responses that contribute to amyloidogenesis and inflammatory neurodegeneration characteristic of the AMD process.

Human Retinal Tissues
Human post-mortem retinal tissues and/or total RNA and/or total protein extracts from human retina were obtained from archived material at the LSU Neuroscience Center, New Orleans LA, from the Southern Eye Bank, Metairie LA or from commercial sources; all AMD samples obtained from dry AMD tissues were used in accordance with the institutional review board (IRB)/ethical guidelines at the LSU Health Sciences Center where these studies were approved [22]. The mean age +/-one standard deviation of the control and AMD retinal tissues were 70.1+/-8.5 and 72.3/-7.5 years respectively, the age range was 65-78 years for all tissues, and all retinal samples were from female Caucasians. Because post-mortem interval (PMI) is a factor that can affect RNA stability and quality, all RNAs were derived from tissues having a PMI of 2.5 hrs; the RNA A 260/280 ranged between 2.04 and 2.14 for all samples; the RNA integrity number for each sample was 8.5 or greater (Fig 1) [19,20,22,23,[25][26][27].

Statistical analysis of data and interpretation
An unchanging miRNA-183 and the abundant 107 nucleotide 5S ribosomal RNA (5SRNA) marker were used as non-coding ssRNA internal controls for miRNA determinations. Relative miRNA and TREM2 mRNA signal strengths were quantified against miRNA-183 and/or 5SRNA in each sample using data-acquisition software provided with a GS250 molecular imager (Bio-Rad, Hercules, CA). Graphic presentations were performed using Excel algorithms (Microsoft, Seattle, WA) and Adobe Photoshop 6.0 (Adobe Systems, San Jose CA). In this paper statistical significance was analyzed using either a Student's t-test or a two-way factorial analysis of variance (p, ANOVA; SAS Institute, Cary, NC). A p<0.05 was deemed as statistically significant; all experimental values were expressed as means +/-one standard deviation (SD) of that mean (Figs 1 and 3

Results
Human retinal tissues-case selection and total RNA quality PMIs for age-matched control or AMD human retinal tissues were all 2.5 hr; age-matched control or AMD sample tissues exhibited no significant differences in age, PMI, RNA A 260/280 indices or RNA integrity numbers (RIN), age-matched control versus AMD. We also noted no significant differences in total RNA purity or yields between the control and AMD groups for any tissues analyzed in this study.
Relative abundance of miRNA-34a and TREM2 in control and AMD retina miRNA arrays were initially used to screen for relative miRNA abundance in adult control whole retina, and then quantitative differences between adult control and AMD miRNA in the the macular region were further examined [20,26] (Fig 1A-1C). Similar to previous reports of total miRNA abundance showed relative abundance in control retina was 5SRNA>>miRNA-125b>>miRNA-34a>>miRNA-146a>>miRNA-155>>miRNA-183 both in adult control and AMD retinal tissue samples. However, miRNA-34a in the retina exhibited up to a 6.3-fold increases AMD over control in advanced AMD. Although relatively abundant in the human retina, 5SRNA, and the much less abundant miRNA-183 exhibited no significant change in relative abundance among any of the samples tested. Of the 5 miRNAs found to be significantly up-regulated on these panels (Fig 1) miRNA-34a and miRNA-146a showed the greatest upregulation in AMD ranging between 2.1-to 6.3-fold over age-matched controls (p<0.01, ANOVA). Interestingly all these 5 miRNAs have been extensively studied and previously categorized as being inducible and regulated by the pro-inflammatory transcription factor NF-kB [14,16,19,30]. It should be further noted that other miRNAs besides miRNA-34a (and miRNA-146a) may participate in regulating the expression of TREM2 at the post-transcriptional level.

TREM2 is down-regulated in AMD retina
Human post-mortem retinal tissues were analyzed as 'control whole retina' and 'macular region' and 'AMD whole retina and macular region' and TREM2 protein abundance was assayed using Western blot analysis and/or ELISA (data not shown). At the level of protein TREM2 was found to be significantly down-regulated in AMD retina in both whole retina and the macular region to respectively 0.54-and 0.22-fold of controls (Fig 1D-1F; data not shown); hence both miRNA-34a increases and TREM2 deficits were found to be co-localized within the same retinal sample (Fig 1A-1C).
Bioinformatics and complementarity analysis for the miRNA-34a-TREM2-mRNA-3'UTR interaction Chromosomal assignments, gene structural schematics and DNA sequences obtained from publically accessible databanks, previously published reports or derived from DNA sequence analysis of human chromosomes 1p and 6p are further described in Fig 2. As indicated in Fig  2 the controlled expression of TREM2 appears to be orchestrated by both transcriptional and post-transcriptional processes originating on 2 separate chromosomes: hsa-miRNA-34a encoded at chr1p36.15 and TREM2 is encoded at human chr6p21.1 [7,8,12,[36][37][38][39][40].

Relative abundance of TREM2 in control and stressed murine MG cells
Stressed MG cells have been used to study mechanistic aspects of inflammatory gene expression and innate-immune signaling in in vitro models of human CNS neurodegeneration, chelation and metal-induced neurotoxicity [12,19,21,[41][42][43]. When compared to control MG cells at 3 days of culture, MG cells treated with TNFα alone showed a 2.1-fold decrease in TREM2 signal; MG cells treated with a negative control miRNA-34a (miRNA-NC) sequence show no significant deficits in TREM2 signal compared to control (Fig 3A and 3B). In contrast MG cells treated with a LNC-stabilized miRNA-34a sequence exhibited a significant 2.3-fold decrease in TREM2 signal (Fig 3B). Results from a typical Western blot analysis are shown for TREM2 and DAP12 abundance versus β-actin abundance in the same sample (upper panel), and in bar graph format (lower panel) (Fig 3B). Results are consistent with a TNFα-or miRNA-34a mediated down-regulation in the expression of TREM2 with no effects on the expression of DAP12. Importantly, as additional controls, a scrambled (SC) miRNA-34a sequence, other 'pro-inflammatory miRNAs such as miRNA-125b, miRNA-146a or miRNA-183 showed no such significant interactive effects (Figs 3B and 4); (data not shown). Fig  4; briefly, transfection of MG cells with a pLightSwitch TREM2-3'UTR-luciferase reporter (containing the entire 299 nt TREM2 mRNA 3'UTR; showed no significant effects on relative luciferase signal strength in controls, however, in the presence of a miRNA-34a mimic TREM2-3'UTR-luciferase signals were quenched~4.5-fold of control and the results were highly significant (Fig 4). The scrambled control miRNA-34a-sc or miRNA-183 exhibited no effects in miRNA-34a-TREM2-3'UTR transfected MG cells. In agreement with previous reports, these data again suggest a productive miRNA-34a-mediated TREM2 mRNA interaction and a miRNA-34a-mediated down-regulation of TREM2 expression [10,12,21,[44][45][46][47][48][49][50][51][52][53].

Functional validation of a miRNA-34a-TREM2-3'UTR interaction in MG cells is shown in
miRNA-34a and TREM2 abundance in ROS-, IL-1β-or TNFα-stressed MG cells and treatment with PBN, CAY10512, CAPE or curcumin ROS-, IL-1β-or TNFα stressed MG cells were found to induce miRNA-34a levels to 1.8-, 1.65and 2.45-fold, respectively, over control levels while reducing TREM2 protein to 0.75-, 0.65and 0.51-fold of control levels in the same sample (Fig 5). Because TNFα exhibited the strongest induction of miRNA-34a, the TNFα-mediated induction in murine MG cells was studied further. The use of the free radical scavenger PBN or the NF-kB inhibitors CAY10512, CAPE or curcumin were each found to lower ROS-, IL-1β-or TNFα-mediated inducibility of miRNA-34a while restoring TREM2 protein back to 0.85-, 0.72-, 0.88-and 0.76-fold of control levels, respectively (Fig 5). Interestingly, CAPE, an active component of propolis from honeybee hives and known to have anti-carcinogenic, anti-mitogenic, anti-inflammatory and immune-modulatory properties was found to be the most effective NF-kB inhibitor of TNFαinduced miRNA-34a used in these studies (Fig 5) [30,41,[54][55][56].

Phagocytosis of Aβ42 peptides; impairment in the presence of insufficient TREM2
We next studied the ability of MG cells to ingest or 'phagocytose' freshly prepared Aβ42 peptide monomers under various conditions in which miRNA-34a and TREM2 levels were altered (Fig 6). Fig 6 (top row, panel A-D) shows staining for Aβ peptides, TREM2, DAPI and a merged view suggesting that that MG cells efficiently internalized Aβ42 peptides from the extracellular medium (yellow merge; λ max~5 80 nm; top row panel D). Briefly, stained sections displaying the intracellular Aβ42 in miRNA-34a treated or control MG cells were quantified for immunofluorescent intensity using SlideBook 5.0 and ImageJ software (NIH) as previously described [44]. The inclusion of a scrambled miRNA-34a sequence (miRNA-34a-sc; or other miRNAs, data not shown) had no significant effect on this process (middle row, panel A-D). However in the presence of a miRNA-34a mimic we observed: (i) significant decreases in the TREM2 signal (Fig 6; lowest row, panel B); (ii) a significantly reduced internalization of the Aβ42 peptides; and (iii) an increased amount of Aβ42 peptides that remained in the extracellular space (Fig 6; lower row, panel D).

Discussion
Amyloidogenesis, the progressive deposition of pro-inflammatory Aβ peptides, is a prominent feature of several age-related neurological diseases of the human CNS including AMD. The dense, insoluble, pro-inflammatory lipoprotein-enriched lesions of AMD called drusen contain abundant βAPP-derived Aβ42 peptides at their core [1][2][3]. Via tandem beta-and gammasecretase cleavage Aβ42 peptides are initially generated as monomers from βAPP, however due largely to their intensely lipophilic-hydrophobic character (21.4% valine-isoleucine) these monomers self-aggregate into higher order structures including dimers, oligomers and fibrils. These aggregates ultimately contribute to the formation of drusen that in part characterize the dry form of AMD [2][3][4][5][31][32][33][34][35][36]. The CNS and retina have evolved highly effective MG cellmediated clearance mechanisms to phagocytose Aβ42 peptide monomers from the extracellular space using transmembrane-associated glycoprotein sensors such as TREM2. When Aβ42 peptide monomers are overproduced, or if deficits in TREM2 and other components of the phagocytic system fail to clear Aβ42, amyloids accumulate and self-aggregate with pro-inflammatory and pathological consequences [6][7][8][9][10][37][38][39][40][41]. Interestingly, the transition of Aβ42 peptide monomers into higher-order structures may be especially stimulated in the presence of physiologically realistic (low nanomolar) concentrations of neurotoxic metals including metal sulfates, such as aluminum sulfate [42,43]. Deficits in other immunoglobulin-like MG transmembrane glycoproteins, including the 67 kD CD33/Siglec-3 (sialic acid-binding immunoglobulin-like lectin-3; gp67; chr 19q13.3) sialoadhesion protein have also been recently described as contributing to the impairment in Aβ42 peptide clearance from the brain, and decreased expression of CD33/Siglec-3 has recently been reported in the peripheral mononuclear cells of AD patients [45].
microRNAs are a fascinating class of small, non-coding 21-25 nt ssRNAs and are the smallest carriers of highly specific genetic information so far discovered, remarkably similar in structure and pathogenic mechanism to ssRNA viroids that cause unusual progressive degenerative diseases in plants [54][55][56][57][58]). The major mechanism of miRNA actions are to recognize complementary RNA sequences in the 3'UTR of their target mRNAs and down-regulate or quench expression of that target mRNA (Figs 2 and 4) [55][56][57][58]. Several inducible miRNAs such as miRNA-34a are under transcriptional control by the pro-inflammatory transcription factor NF-kB in the CNS and other tissues, so up-regulated NF-kB, via increasing specific miRNA abundances, may ultimately act as an important down-regulator of the expression of multiple sporadic AMD-relevant genes [22,25,29,56,58,59]. In this study we initially characterized total miRNA expression in whole AMD retina, in a macular-enriched region of the AMD retina, and in ROS-(peroxide), IL-1β-and/or Aβ42-treated MG cells and found a significant up-regulation of an NF-kB-sensitive miRNA-34a closely linked to a down-regulation in a miRNA-34a mRNA target encoding TREM2 within the same tissue and cell samples. Specific up-regulation of miRNA-34a-signalling has been previously associated with at least 15 neurological, neuroimmune, neuro-inflammatory and/or neurodegenerative pathologies including (i) diseased spinal cord tissues in amyotrophic lateral sclerosis [11]; (ii) peripheral blood mononuclear cells and blood plasma in sporadic AD patients [60,61]; (iii) altered immunological signaling associated with multiple sclerosis [62]; (iv) autoimmune encephalomyelitis [37,38]; (v) progressive neurotrophic deficits, including dysfunctional Bcl-2 signaling, in transgenic murine models of AD (TgAD) including the APPswe/PSDeltaE9 model [63]; (vi) altered synaptotagmin-1 and syntaxin-1A signaling, synaptogenesis and neurite outgrowth [64]; (vii) repression in the expression of several genes involved in cell survival and oxidative defense pathways such as Bcl2 and SIRT1 [61]; (viii) accelerated aging of the murine brain [65]; (ix) deficient immune-and phagocytoticresponses in progressive inflammatory degeneration in cardiovascular disease [66]; (x) aging of the vasculature and cellular senescence [66][67][68]; (xi); the mis-regulation of p53-regulated genes contributing to DNA damage, p53-mediated apoptosis and mitotic catastrophe [69]; (xii) astroglial cell proliferation, gliosis and tumor progression [70]; (xiii) lower mini-mental state examination (MMSE) scores linked to elevated miRNA-34a in the blood plasma of AD patients [61]; (xiv) major depressive disorder (MDD) [67]; and (xv) progressive inflammatory neurodegeneration and epileptiform activities associated with epilepsy and the early stages of AD [12,71]. The involvement of miRNA-34a in epilepsy and AD is particularly interesting because of the overlapping neuropathology of these two disorders with respect to seizure frequency and cognitive decline first apparent in the earliest stages of each disease [70][71][72].
The current work provides at least 8 lines of evidence to suggest that an NF-κB-sensitive miRNA-34a acts as a repressor of its TREM2 mRNA target: (i) up-regulation of miRNA-34a in AMD retina corresponds to down-regulation of TREM2 in the same retinal tissues and stressed MG cells (Figs 1, 2, 3 and 4); (ii) human miRNA-34a is highly complementary to the human TREM2 mRNA 3'UTR (~88% homology between the miRNA-34a 5' region and the TREM2-3'UTR seed sequence; structural stability~-22 kcal/mol; Figs 2 and 4); (iii) when a stabilized miRNA-34a, but not negative control (NC) or scrambled (SC) miRNAs are added to MG cells in culture there is a significant down-regulation of TREM2 signal (Fig 3); (iv) ROS, IL-1β-and TNFα-stressed MG cells exhibit up-regulation of miRNA-34a corresponding to down-regulation of TREM2 in the same sample ( Fig 5); (v) the free radical scavenger PBN reduces TNFαmediated up-regulation of miRNA-34a and significantly restores TREM2 levels (Figs 3 and 5); (vi) the substituted trans-stilbene resveratrol analog and NF-κB inhibitors CAPE, CAY10512 and curcumin also strongly inhibited miRNA-34a promoter-luciferase reporter activity (Figs 4 and 5); (vii) an anti-miRNA-34a (AM34a) specifically directed against miRNA-34a was found to restore TREM2 protein to near control levels in stressed MG cells ( Fig 5); and (viii) an exogenously added miRNA-34a mimic (but not NC or SC miRNAs) were found to down-regulate TREM2 and impair MG cells from phagocytosing Aβ42 peptide monomers (Fig 6). Taken together the results suggest involvement of an oxidative stress-and NF-kB-inducible miRNA-34a in the regulation of TREM2 and TREM2-mediated phagocytic activities in the CNS. Indeed the contribution of NF-kB and the potential use of NF-kB inhibitors in managing the up-regulation of NF-kB-sensitive pro-inflammatory miRNAs with pathological consequences has been extensively addessed in several recent reports [10,12,20,41,[56][57][58][59]. Although we examined miRNA-34a and TREM2 levels in 21 carefully selected human sporadic AMD and agematched control retina other miRNAs or other genetic factors may play ancillary roles in phagocytosis in this retinal disease, especially in diverse human population sets [73][74][75]. MG cell age may be a variable factor with older MG cells exhibiting reduced immune responses and phagocytic capabilities (unpublished observations) [19][20][21][22]. At this point while the NF-kB-miRNA-34a-TREM2 circuit appears to be dysfunctional in AMD retina and in ROS-and cytokine-stressed MG cells we cannot exclude that other brain-enriched miRNAs have ancillary control on TREM2 mRNA stability and expression in the retina and CNS.
In summary, dysfunctional phagocytosis in AMD retina and Aβ42 peptide aggregation into drusen have been attributed to disease-specific deficits in Aβ42 clearance. These current data are the first to suggest that the pathological up-regulation of miRNA-34a abundance in AMD retina and in stressed MG cells are linked to a functional repression of TREM2 expression and bioavailability. The results further suggest that the mis-regulation of specific miRNAs in AMD contributes to amyloidogenesis, a known driver of inflammatory neuropathology and disruption of normal innate-immune and inflammatory responses. It appears that Aβ42 peptide generation and clearance are a carefully orchestrated, and continually maintained homeostatic mechanism, in the CNS that when upset leads to amyloidogenic and pathological consequences. While healthy MG appear to deal relatively easily with Aβ42 peptide monomers they appear to encounter difficulty: (i) when Aβ42 peptide monomers are generated in excess, or (ii) when amyloid dimers and higher order oligomeric Aβ42 peptide structures preclude TREM2-mediated phagocytosis. This further suggests that Aβ42 monomer phagocytosis is in a steady state and that any factors that promote aggregation of Aβ42 monomers, including excessive Aβ42 peptide generation, or coalescence via neurotoxic metals or other factors, may easily upset this sensitive homeostatic balance. Fig 7 is a highly schematicized depiction of the actions of an NF-kB-regulated, miRNA-34a-mediated TREM2 sensor-phagocytosis system down-regulated in stressed MG cells and AMD. Signaling via the TREM2/tyrosine binding protein/ DNAX-activating protein of 12 kDa (TYROBP/DAP12) receptor complex results in phagocytosis and ultimately, clearance of Aβ42 peptides from the extracellular medium [13,[76][77][78][79][80][81]. Interestingly, TREM2 knockdown or knockout mice exhibit attenuated immunological responses and/or increases in age-related neuroinflammatory biomarkers [79,80]. It has also recently been shown that TREM2 deficiencies exacerbate tau pathology and promote neurodegenerative change and spatial learning deficits in P301S tau transgenic mice [81]. Importantly no deficits in the TYROBP/DAP12 adaptor protein have been observed in stressed MG cells or in AMD retina (Fig 3; unpublished observations).
Lastly, using multiple interdependent techniques it should be mentioned that miRNA-34a has also been recently found to be significantly increased in limbic regions of the AD-affected brain, in AD monocytes and in several amyloid overexpressing transgenic murine models of AD (unpublished observations) [10,12,48,60,61,63,65]. A dysfunctional TREM2 sensor-receptor, through a loss-of-function mutation in familial AD, may have the same net end result as an insufficient amount of a functional TREM2 phagocytosis-sensor with both pathological scenarios resulting in a significant impairment in the ability to effectively phagocytose and clear Aβ42 peptides. The data reported here are the first to indicate that the orchestrated interaction of at least two independent gene products on two different human chromosomes-miRNA-34a at chr1p36.22 and TREM2 at chr6p21.1-are required to modulate TREM2 activities, the sensing of potentially hazardous amyloidogenic molecules in the extracellular space, and the phagocytosis and clearance of retinotoxic debris to maintain functional homeostasis in the retina. Notably, this type of multigenic miRNA-34a-mediated regulation would escape detection by standard GWAS-based genomic analysis. Because miRNA-34a is encoded as an NF-kB-sensitive transcript, anti-NF-kB and/or anti-miRNA strategies and/or combinatorial approaches, perhaps with other targeted anti-inflammatory therapies, may be useful in the clinical management of AMD and in other disorders of the CNS with an amyloidogenic component [79][80][81][82][83][84][85].