Presenilin 2 influences miR146 level and activity in microglia

Abstract

Microglia, the resident innate immune cells of the CNS, are the primary defenders against microbes and critical to CNS remodeling. Dysregulation of microglial behavior can lead to unchecked pro-inflammatory activity and subsequent neurodegeneration. The molecular mechanisms leading to chronic inflammation and microglial dysfunction in neurodegenerative diseases are not well-understood. It is known that patients with Presenilin 2 (PS2) mutations develop autosomal dominant Alzheimer disease. We have shown that a lack of normal PS2 function is associated with exaggerated microglia pro-inflammatory responses in vitro. To identify pathways by which PS2 regulates microglia and determine how PS2 dysfunction may lead to altered inflammatory pathways, we pursued an unbiased array approach to assess differential expression of microRNAs between murine PS2 knockout (KO) and wild-type microglia. We identified miR146, a negative regulator of monocyte pro-inflammatory response, as constitutively down-regulated in PS2 KO microglia. Consistent with a state of miR146 suppression, we found that PS2 KO microglia express higher levels of the miR146 target protein interleukin-1 receptor-associated kinase-1, and have increased NFκB transcriptional activity. We hypothesize that PS2 impacts microglial responses through modulation of miR146a. PS2 dysfunction, through aging or mutation, may contribute to neurodegeneration by influencing the pro-inflammatory behavior of microglia. Presenilin 2 (PS2), a membrane associated protease, has been implicated in the pathogenesis of Alzheimer disease. We have previously shown that PS2 plays an important role in curbing the proinflammatory response in microglia. Here, we report the novel finding that PS2 participates in maintaining the basal and cytokine induced expression of the innate immunity regulating microRNA, miR146. These data suggest one mechanism by which PS2 works to reign in proinflammatory microglial behavior and that PS2 dysfunction or deficiency could thus result in unchecked proinflammatory activation contributing to neurodegeneration.

Keywords: Alzheimer disease; miR146; microglia; neuroinflammation; presenilin.

Fig. 1

Presenilin 2 (PS2) knockout (KO) primary microglia have suppressed expression and induction of miR146a. (a) RNA collected from primary PS2 KO or wild-type microglia was assayed for miR146a transcript by RT-PCR. Levels of miR146a are significantly decreased in PS2 KO microglia. (n = 4, *p < 0.05). RT-PCR for miR146 transcript in RNA collected from wild-type and PS2KO microglia after 24 h incubation with (b) 10 μ/mL IFNγ or (c) 100 ng/mL lipopolysaccharide (LPS) reveals a significant difference between WT and PS2KO microglia in the induction of miR146 by IFNγ but not LPS (n = 4 for each set of data, *p < 0.05).

Fig. 2

MiR146a target, interleukin-1 receptor-associated kinase-1 (IRAK-1) is increased in presenilin 2 (PS2) knockout (KO) microglia. (a) Protein lysates prepared from wild-type and PS2 KO microglia were analyzed by Western blot for expression of IRAK-1 protein, (representative immunoblot shown). Densitometry from four independent experiments is graphed demonstrating that IRAK-1 is significantly increased in PS2KO microglia. (b) IRAK-1 levels measured by western blot after miR146 over-expression in PS2KO microglia are significantly decreased compared to PS2KO microglia over-expressing scrambled control sequence (*p < 0.05).

Fig. 3

NFκB transcriptional activity is enhanced in presenilin 2 (PS2) knockout (KO) microglia in response to a Toll-like receptor 4 (TLR4) agonist. NFκB activity in PS2 KO or wild-type microglia was measured by a luciferase reporter assay. PS2 KO microglia show increased NFκB activity compared to wild type (n = 5, *p < 0.05).