Reduced miR-659-3p Levels Correlate with Progranulin Increase in Hypoxic Conditions: Implications for Frontotemporal Dementia

Abstract

Progranulin (PGRN) is a secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated microglia. Loss-of-function mutations in the GRN gene are an important cause of familial frontotemporal lobar degeneration (FTLD). PGRN has a neurotrophic and anti-inflammatory activity, and it is neuroprotective in several injury conditions, such as oxygen or glucose deprivation, oxidative injury, and hypoxic stress. Indeed, we have previously demonstrated that hypoxia induces the up-regulation of GRN transcripts. Several studies have shown microRNAs (miRNAs) involvement in hypoxia. Moreover, in FTLD patients with a genetic variant of GRN (rs5848), the reinforcement of miR-659-3p binding site has been suggested to be a risk factor. Here, we report that miR-659-3p interacts directly with GRN 3′UTR as shown by luciferase assay in HeLa cells and ELISA and Western Blot analysis in HeLa and Kelly cells. Moreover, we demonstrate the physical binding between GRN mRNA and miR-659-3p employing a miRNA capture-affinity technology in SK-N-BE and Kelly cells. In order to study miRNAs involvement in hypoxia-mediated up-regulation of GRN, we evaluated miR-659-3p levels in SK-N-BE cells after 24 h of hypoxic treatment, finding them inversely correlated to GRN transcripts. Furthermore, we analyzed an animal model of asphyxia, finding that GRN mRNA levels increased at post-natal day (pnd) 1 and pnd 4 in rat cortices subjected to asphyxia in comparison to control rats and miR-659-3p decreased at pnd 4 just when GRN reached the highest levels. Our results demonstrate the interaction between miR-659-3p and GRN transcript and the involvement of miR-659-3p in GRN up-regulation mediated by hypoxic/ischemic insults.

Progranulin and Its Related MicroRNAs after Status Epilepticus: Possible Mechanisms of Neuroprotection

“…The same workgroup already demonstrated before, that hypoxic conditions in cells cause an increase of PGRN expression [21]. In their more recent paper this theory has been confirmed in a rat model [22]. It has also shown that miR-659-3p decreases under hypoxic conditions while the concentration of PGRN increases with a delay, probably similar to the PGRN levels seen in humans and rats.…”

“…PGRN has a neuroprotective role via promoting neurite outgrowth in neurons [16,17], and as a promoter of neuronal survival via Sortilin and P75 NTR [7,18,19,20] It also curbs the cytokine expression by CD11b positive macrophages and CD11b positive microglia [11]. PGRN expression in the CNS is elevated following hypoxia [21,22], neuroinflammation [23], trauma [24,25,26], stroke [27] and status epilepticus [5,6]. Up until now three PGRN-receptors have been identified, each with different functions.…”

“…Under hypoxic conditions, PGRN and microRNAs (miRNA) possibly controlling PGRN have been shown to be upregulated in neuroblastoma cells [22]. When measuring PGRN messengerRNA after a hypoxic stress, a delayed increase with a peak at Day 4 was seen [22].…”

“…When measuring PGRN messengerRNA after a hypoxic stress, a delayed increase with a peak at Day 4 was seen [22]. This delay could be due to neuroprotective and anti-inflammatory mechanisms mediated partly by the PGRN pathway.…”

“…For FTD, this happens to be PGRN. Since the majority of miRNA control genes through modification at the 3′UTR-end, Piscobo et al [22] analyzed the 3′UTR-end of the GRN -gene and found two possible binding sites for miR-659-3p. Further tests showed that an increase of miRNA-659-3p caused a decreased expression of GRN-RNA, this suggests that binding of miRNA-659-3p with the GRN-RNA inhibits its translation.…”