text:
miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1)
Jaekwang Kim1,
Fabienne C. Fiesel1,
Krystal C. Belmonte1,
Roman Hudec1,
Wang-Xia Wang2,
Chaeyoung Kim1,
Peter T. Nelson2,
Wolfdieter Springer1,3 &
Jungsu Kim ORCID: orcid.org/0000-0002-6931-85811,3
Loss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson's disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels.
Here, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3′-untranslated region (3′UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b.
We demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.
Parkinson's disease (PD), the second most common neurodegenerative disorder, is clinically manifested by motor symptoms, such as bradykinesia, resting tremor, rigidity, and postural instability [1]. PD is pathologically characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SN) [1]. Identification of several genetic risk factors has led to a significant advance in our understanding of PD pathogenesis. To date, mutations in several genes, such as SNCA (α-synuclein), Leucine-rich repeat kinase 2 (LRRK2), DJ-1, PARK2 (PARKIN), and PTEN-induced putative kinase 1 (PINK1) [1], have been shown to cause familial early-onset PD, accounting for 5–10 % of all cases [2].
Mounting evidence suggests mitochondrial dysfunction as a central mechanism in PD pathogenesis [3]. For instance, mitochondrial toxins, such as MPTP and rotenone, induce parkinsonism in animal models and human [4]. Moreover, damage to mitochondrial DNA in SN is increased in aging and PD patients [5, 6]. Furthermore, the activity of complex I is reduced in SN of PD patients [7, 8]. Therefore, a better understanding of how the integrity and function of mitochondria are maintained following insults may provide novel opportunities for PD treatment.
Mutations in PARKIN and PINK1 are the leading causes of autosomal recessive PD [9, 10]. Together, they functionally mediate the stress-activated, selective clearance of impaired mitochondria via autophagic degradation (termed mitophagy) [11], supporting the idea that deficits in mitochondria quality control may be a core mechanism underlying PD pathogenesis. PINK1, a mitochondrial serine/threonine kinase, is maintained at low levels under normal physiological conditions. Upon mitochondrial damage, PINK1 is quickly stabilized on damaged mitochondria, which leads to translocation of cytosolic Parkin to damaged mitochondria [11–13]. PINK1 activates Parkin by phosphorylating it at Ser65. Once activated, Parkin, an E3 ubiquitin ligase, selectively tags the damaged mitochondria for mitophagic clearance by ubiquitinating multiple mitochondrial proteins [14, 15]. Recently, several groups discovered that PINK1 also phosphorylates ubiquitin at Ser65 (pUbS65) and PINK1-dependent phosphorylation of ubiquitin is required for translocation and activation of Parkin to initiate mitophagy [16–18]. Moreover, pUbS65 has been suggested to accumulate with mitochondrial stress, age, and disease [19] and to serve as a specific signal for recognition by autophagy receptors [20, 21]. Therefore, accumulation of pUbS65 is a hallmark of mitochondrial stress/damage. Beyond their roles in mitophagy, mounting evidence suggested that both PINK1 and Parkin have neuroprotective roles against various insults, such as oxidative stress and PD-relevant toxins, although the underlying mechanisms still remain elusive [22, 23].
Given its critical roles in mitochondrial homeostasis and neuroprotection, the mechanism of how PINK1 expression is regulated has been under intense investigations. Although PINK1 regulation at the post-translational level has been extensively studied [24], little is known about the regulation mechanism of PINK1 at transcriptional or translational levels. microRNAs (miRNAs) have been gaining growing attention as key regulators of protein coding genes [25, 26]. Small non-coding miRNAs, ~22 nucleotides, are generated from primary transcripts via sequential cleavages by Drosha, ribonuclease type III (DROSHA) and Dicer 1, ribonuclease type III (DICER1) complexes [27]. miRNAs regulate expression of their target genes by binding to cognate messenger RNAs (mRNAs), leading to translational repression [27]. Thus far, a few miRNAs are identified as regulators of PD-associated genes, such as SNCA, LRRK2, and DJ-1 [28–31]. However, post-transcriptional regulation of human PINK1 by miRNAs is poorly defined.
Here, we identified a novel regulatory mechanism of PINK1 by miR-27a/b via targeting its 3′UTR. We demonstrated that miR-27a and miR-27b inhibit PINK1 stabilization, thereby preventing autophagic degradation of impaired mitochondria.
PINK1 expression is regulated by miRNAs
PINK1 mRNA (NM_032409) has a long 3′UTR, ~840 nucleotides, raising the possibility that its expression may be regulated by miRNAs which often target 3′UTR sequences. The mature miRNAs bind to their target mRNAs through incorporation into the RNA-induced silencing complex (RISC) which is composed of Argonaute (AGO) and several other partners [32]. To determine if PINK1 expression is regulated by miRNAs, we first assessed whether human PINK1 mRNA is associated with RISC complex using AGO-specific RNA immunoprecipitation method in HeLa cells (Fig. 1a). AGO2-bound miRNA/mRNA complexes were first pulled down with an AGO-specific antibody. Non-specific IgG served as negative control. PINK1 mRNA levels were then analyzed by qRT-PCR. Pull-down with anti-AGO antibody resulted in dramatic increase of PINK1 mRNA levels compared to the control IgG, whereas AGO2 knock-down with siRNA markedly decreased PINK1 mRNA levels pulled down by anti-AGO antibody (Fig. 1b, c). We next tested whether inhibition of miRNA biogenesis via DICER1 knock-down would affect PINK1 protein levels. As expected, DICER1 knock-down decreased miRNA levels in HeLa cells (Additional file 1). Compared to the negative control, DICER1 knock-down with siRNA significantly increased PINK1 protein levels in HeLa cells (Fig. 1d, e). Collectively, our data indicate that human PINK1 expression is regulated by miRNAs.
Regulation of PINK1 by miRNA. a Schematic diagram of AGO-specific RNA immunoprecipitation assay. b, c PINK1 mRNAs are associated with miRNA/RISC complex. HeLa cells were transfected with 50 nM of AGO2 siRNA or negative control. 48 h post-transfection, AGO2-bound mRNAs were pulled down from cell lysates with 2A8 anti-AGO2 antibody or non-specific IgG antibody. PINK1 mRNA levels were then measured by qRT-PCR. siRNA against AGO2 served as a quality control for AGO-specific RNA immunoprecipitation assay. PINK1 mRNA levels were quantified as a percentage of group 1 (n = 6, two-way ANOVA). d, e Knock-down of DICER1 increased PINK1 protein levels. HeLa cells were transfected with 50 nM DICER1 siRNA (DICER1 KD) or negative control (Ctl). 48 h post-transfection, cells were harvested for Western blot. The arrowhead indicates a full-length PINK1 band and the asterisk indicates a non-specific band. Each protein level was normalized to corresponding GAPDH level and quantified as a percentage of control (n = 4, t-test). Values are mean ± SEM (n.s. = non-significant, **p < 0.01, ***p < 0.001)
miR-27a/b suppress human PINK1 expression by directly targeting 3′UTR of its mRNA
To identify potential miRNAs that directly regulate PINK1 expression, we first searched miRNAs that have putative binding sites in the 3′UTR of human PINK1 mRNA by utilizing several miRNA-target prediction algorithms as described previously [33]. Among several candidates, we focused on miR-27a and miR-27b (hereafter referred to as miR-27a/b) because 1) miR-27a/b are commonly predicted by several algorithms to have seed match sequences in the 3′UTR of human PINK1 mRNA (Fig. 2a and Additional file 2a), 2) miR-27a/b are expressed in SN of human midbrain (Additional file 2a) [34–36], 3) miR-27a/b have multiple putative binding sites in the 3′UTR of human PINK1 mRNA with low binding free energies (Additional file 2b) [37]. Of note, both putative binding sites of miR-27a/b in the 3′UTR of PINK1 mRNA are well conserved in primates, such as human, chimpanzee, and monkey, but not in rodents, such as rat and mouse (Additional file 3), raising the possibility of primate-specific regulation of PINK1 expression by miR-27a/b.
miR-27a/b suppress human PINK1 expression. a Schematic diagram of the conserved target sites of miR-27a/b in the 3′UTR of human PINK1 mRNA. Seed sequences were indicated in red. Asterisks (*) indicate the conserved nucleotides. b Schematic diagram of the luciferase reporter plasmids. The reporter constructs contain the full-length 3′UTR of human PINK1 mRNA with wild-type (WT) or mutated (Mut) seed match sites downstream of Renilla luciferase gene. c Overexpression of miR-27a/b decreased luciferase activities in HeLa cells. Cells were transfected with miR-27a/b or negative control (Ctl) along with reporter constructs as indicated. 48 h post-transfection, luciferase activities were measured (n = 4, one-way ANOVA). Renilla luciferase activity was normalized to the corresponding firefly luciferase activity. d-g Overexpression of miR-27a/b decreased PINK1 protein levels in HeLa (D-E) and M17 cells (F-G). Cells were transfected with miR-27a/b or negative control (Ctl). 48 h post-transfection, cells were harvested for Western blot (n = 4, t-test). PINK1 protein level was normalized to corresponding GAPDH level and quantified as a percentage of control. Data are shown as a percentage of control. Values are mean ± SEM (n.s. = non-significant, **p < 0.01, ***p < 0.001)
To determine if miR-27a/b directly suppress PINK1 expression by targeting its 3′UTR, we performed a luciferase assay with the reporter construct containing the entire 3′UTR of human PINK1 mRNA downstream of luciferase (Fig. 2b). miR-27a/b significantly suppressed the expression of luciferase in HeLa cells (Fig. 2c). Mutations in either seed match site1 or site2 significantly increased luciferase activities compared to wild-type (Fig. 2c). Moreover, a combined mutation in both seed match sites (Mut 1&2) completely abolished the miR-27a/b-mediated reduction of luciferase activities, indicating that both seed match sites are functional (Fig. 2c). There was no difference in the levels of miR-27a and miR-27b expressed between the miR transfected groups (Additional file 4). To determine if miR-27a/b affect PINK1 protein levels, we first transfected synthetic miR-27a, miR-27b, or negative control to human HeLa cells (Additional file 5a). Overexpression of miR-27a/b markedly decreased PINK1 protein levels in HeLa cells (Fig. 2d, e). A combination of miR-27a and miR-27b did not further decrease PINK1 levels in HeLa cells, suggesting that both miR-27a and miR-27b target the same site in the 3′UTR of PINK1 mRNA (Additional file 6). To evaluate the effect of miR-27a/b in another cell type, we tested M17 cells. miR-27a/b also suppressed PINK1 expression in human dopaminergic-like M17 cells (Fig. 2f, g).
Next, we examined whether endogenous miR-27a/b regulate PINK1 expression. To inhibit miR-27a/b function, we used locked nucleic acid (LNA)-based anti-miR that is nuclease-resistant single-stranded antisense oligonucleotides complementary to their target miRNAs [38]. Due to the high similarity in the mature sequences of miR-27a and miR-27b, single anti-miR (denoted as anti-miR-27a/b) inhibited both miR-27a and miR-27b (Fig. 3a and Additional file 5b). Inhibition of endogenous miR-27a/b significantly increased luciferase activities from the reporter construct with WT PINK1 3′UTR, whereas combined mutations of both seed match sites blocked the anti-miR-27a/b-mediated induction of luciferase activities in HeLa cells (Fig. 3b). Moreover, Inhibition of endogenous miR-27a/b significantly increased PINK1 protein levels in HeLa cells as well as M17 cells (Fig. 3c-f). Collectively, these data indicate that miR-27a/b suppress human PINK1 expression by directly targeting 3′UTR of its mRNA.
Inhibition of endogenous miR-27a/b function increases human PINK1 expression. a Schematic diagram of sequence alignment of miR-27a/b and anti-miR-27a/b. Asterisks (*) indicate conserved nucleotides. b Inhibition of endogenous miR-27a/b function increased luciferase activities in HeLa cells. Cells were transfected with the reporter constructs containing the full-length 3′UTR of human PINK1 mRNA with wild-type (WT) or mutated (Mut) seed match sites downstream of Renilla luciferase gene. 48 h post-transfection, luciferase activities were measured (n = 4, t-test). c-f Inhibition of endogenous miR-27a/b increased PINK1 protein levels in HeLa (C-D) and M17 cells (E-F). Cells were transfected with 150 nM of anti-miR-27a/b or anti-control (anti-Ctl). 48 h post-transfection, cells were harvested for Western blot. PINK1 protein level was normalized to corresponding GAPDH level and quantified as a percentage of control (n = 4, t-test). Data are shown as a percentage of control. Values are mean ± SEM (n.s. = non-significant, **p < 0.01, ***p < 0.00)
PINK1 translation is indispensable for the accumulation of PINK1 upon mitochondrial damage
PINK1 protein is rapidly and constitutively degraded in healthy mitochondria by mitochondrial proteases, whereas mitochondrial damage allows PINK1 to accumulate on the outer membrane of the damaged mitochondria [39]. However, it has not been well determined whether transcription and/or translation step of PINK1 may contribute to the accumulation of PINK1 protein upon mitochondrial damage. Given a low basal level of PINK1 protein and a rapid build-up of PINK1 protein upon mitochondrial damage, we hypothesized that new synthesis of PINK1 mRNA together with post-translational stabilization may contribute to the accumulation of PINK1 upon mitochondrial damage. Thus, we first examined whether PINK1 mRNA levels are altered by mitochondrial damage. PINK1 mRNA levels were not affected by mitochondrial damages induced by either carbonyl cyanide m-chlorophenylhydrazone (CCCP, mitochondrial uncoupler) or combination of oligomycin (inhibitor of ATP synthase) and antimycin (inhibitor of mitochondrial respiratory chain complex III) in HeLa cells (Fig. 4a). Notably, blocking translation with cycloheximide (CHX) completely abolished the accumulation of PINK1 protein by CCCP (Fig. 4b, c). Taken together, these data suggest that PINK1 translation is critical for the accumulation of PINK1 upon mitochondrial damage.
PINK1 translation is critical for the accumulation of PINK1 upon mitochondrial damage. a PINK1 mRNA levels were not altered by mitochondrial damage. HeLa cells were incubated with 10 μM CCCP or combination of 10 μM oligomycin and 4 μM antimycin for 2 h and then, PINK1 mRNA levels were measured by qRT-PCR. PINK1 mRNA level was normalized to the corresponding GAPDH levels and quantified as a percentage of control (n = 8, t-test). b, c Translation is involved in PINK1 accumulation upon CCCP treatment. HeLa cells were first pre-incubated with 100 μM cycloheximide (CHX) for 2 h and then washed. Cells were further incubated with 10 μM CCCP with or without CHX for 2 h (n = 4, two-way ANOVA). Values are mean ± SEM (n.s. = non-significant, ***p < 0.001)
miR-27a/b inhibit PINK1 accumulation upon mitochondrial damage
Because miR-27a/b suppress PINK1 expression at the translational level, we next examined whether miR-27a/b influence PINK1 accumulation upon mitochondrial damage. miR-27a/b-mediated suppression of PINK1 dramatically reduced the PINK1 accumulation upon CCCP treatment compared to the negative control (Fig. 5a, b), whereas inhibition of miR-27a/b further increased PINK1 accumulation compared to the negative control (Fig. 5c, d). Furthermore, miR-27a/b showed similar effects on PINK1 accumulation upon combined treatment of oligomycin and antimycin (Fig. 5a-d). Taken together, our data suggest that the accumulation of PINK1 upon mitochondrial damage is strongly regulated by expression levels of miR-27a and miR-27b.
miR-27a/b inhibit PINK1 accumulation upon mitochondrial damage. a, b Overexpression of miR-27a/b inhibited PINK1 accumulation upon mitochondrial damage. 48 h post-transfection, HeLa cells were treated with 10 μM CCCP or combination of 10 μM oligomycin and 4 μM antimycin as indicated for 2 h (n = 4, two-way ANOVA). c, d Inhibition of endogenous miR-27a/b increased PINK1 accumulation upon CCCP treatment. (n = 4, two-way ANOVA). PINK1 levels were normalized to corresponding GAPDH level and quantified as a percentage of control. Values are mean ± SEM (n.s. = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001)
miR-27a/b prevent the accumulation of phospho-ubiquitin upon mitochondrial damage
Recent studies demonstrated that PINK1 phosphorylates ubiquitin at Ser65 (pUbS65) upon mitochondrial damage [16–18]. Ubiquitin phosphorylation by PINK1 plays a critical role in the activation and mitochondrial translocation of Parkin [16–18]. We also observed that pUbS65 levels were markedly increased by CCCP and overexpression of PINK1 further increased the pUbS65 accumulation by CCCP in HeLa cells (Fig. 6a). However, overexpression of PINK1 did not affect pUbS65 levels under the basal condition without CCCP treatment (Fig. 6a), suggesting that ubiquitin kinase activity of PINK1 is activated specifically upon mitochondrial damage.
miR-27a/b prevent pUbS65 accumulation upon mitochondrial damage. a, b Overexpression of PINK1 increased pUbS65 accumulation in HeLa cells only when incubated with CCCP. 48 h post-transfection, cells were incubated with 10 μM CCCP for 2 h and pUbS65 levels were determined by Western blot (n = 3, two-way ANOVA). c, d Overexpression of miR-27a/b inhibited pUbS65 accumulation by CCCP in HeLa cells (n = 4, two-way ANOVA). e, f Inhibition of endogenous miR-27a/b increased pUbS65 accumulation by CCCP in HeLa cells (n = 4, two-way ANOVA). PINK1 levels were normalized to corresponding GAPDH level and quantified as a percentage of control. Values are mean ± SEM (n.s. = non-significant, **p < 0.01, ***p < 0.001)
In line with these data, overexpression of miR-27a/b inhibited the accumulation of pUbS65 upon CCCP treatment compared to the negative control (Fig. 6c, d), whereas inhibition of miR-27a/b further increased pUbS65 levels in the presence of CCCP compared to the negative control (Fig. 6e, f). In contrast, pUbS65 levels were not affected by either overexpression or inhibition of miR-27a/b under the basal condition without CCCP treatment (Fig. 6c-f). Taken together, our data demonstrate that miR-27a/b prevent the accumulation of pUbS65 upon mitochondrial damage by suppressing PINK1 expression.
miR-27a/b prevent Parkin translocation to mitochondria upon mitochondrial damage
PINK1-dependent translocation of cytosolic Parkin to damaged mitochondria is a prerequisite for mitophagy [11]. To determine if miR-27a/b prevent Parkin translocation upon mitochondrial damage, we transfected miR-27a/b or negative control to HeLa cells stably expressing Parkin. After inducing mitochondrial damage by CCCP for 2 h, mitochondrial fractions were separated from total cell lysates and then analyzed by Western blot (Fig. 7a). Compared to the negative control, miR-27a/b strongly inhibited Parkin translocation to mitochondria as well as accumulation of active form of LC3 (LC3-II), an autophagic marker, upon CCCP treatment (Fig. 7a, b).
miR-27a/b prevent Parkin translocation to mitochondria upon mitochondrial damage. a, b 48 h post-transfection, HeLa cells were incubated with 10 μM CCCP for 2 h. After isolating the mitochondria-enriched fractions, Parkin translocation and LC3-II were assessed by Western blot. Each protein level was normalized to corresponding mitochondrial loading control (SOD2) level and quantified as a percentage of control (n = 3, two-way ANOVA). Purity of mitochondrial fraction was assessed by monitoring p38 cytosolic and VDAC1 mitochondrial markers. PN; post-nuclear, C; cytoplasm, M; mitochondrial (A). c, d 48 h post-transfection, HeLa cells stably expressing GFP-Parkin were visualized by GFP, a mitochondrial marker (TOM20) antibody, and a nuclear dye (Hoechst) as indicated. Scale bars correspond to 10 μm. Parkin translocation was quantified as the ratio of cytoplasmic to nuclear GFP signal and the resulting ratios were normalized to control. Data were collected from 4 independent replicates (n > 1000 cells) and are shown as a percentage of control. Values are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001)
To confirm our biochemical analysis of miR-27a/b effect on Parkin translocation, we also quantified Parkin translocation by High Content Imaging. After transfected with a negative control or miR-27a/b, HeLa cells expressing GFP-Parkin were treated with CCCP for 2 h, stained with a nuclear dye, and imaged. Parkin translocation was measured by assessing the ratio of cytoplasmic and nuclear GFP signal as described previously [40]. miR-27a/b overexpression markedly decreased the levels of Parkin colocalized with mitochondria marker TOM20, compared to the negative control (Fig. 7c, d). Taken together, our data demonstrate that miR-27a/b prevent the induction of mitophagy by suppressing PINK1 expression.
miR-27a/b inhibit the lysosomal degradation of damaged mitochondria
Damaged mitochondria are selectively tagged by a combination of PINK1 and Parkin and then, delivered to lysosome for hydrolytic degradation [11]. Therefore, we next examined whether miR-27a/b inhibit the delivery of damaged mitochondria to lysosome by using mitochondria-targeting Keima (mtKeima) [41]. Keima is a fluorescent protein derived from coral which is resistant to lysosomal proteases and changes its color upon pH change. mtKeima is engineered to localize in mitochondria and changes its color upon a fusion of neutral autophagosome with acidic lysosome [41]. After transfected with a negative control, PINK1 siRNA, or miRNAs as indicated, HeLa cells expressing mtKeima were treated with CCCP for 12 h and imaged. Lysosomal delivery of damaged mitochondria was measured by assessing the ratio of acidic and neural Keima signal as described previously [41]. As a positive control for the assay, we first tested the effect of PINK1 knockdown. PINK1 knockdown dramatically decreased the lysosomal delivery of damaged mitochondria, validating our assay performance (Additional file 7a, b). miR-27a/b overexpression significantly decreased the lysosomal delivery of damaged mitochondria (Fig. 8a, b). Autophagic clearance of damaged mitochondria was further assessed by monitoring the degradation of COX2, a mitochondrial inner membrane protein. miR-27a/b inhibited the degradation of COX2 upon mitochondrial damage, compared to a negative control miR (Fig. 8c, d). These two independent assays indicate that miR-27a/b inhibit autophagic degradation of damaged mitochondria by lysosome.
miR-27a/b inhibit the degradation of mitochondria via lysosome upon mitochondrial damage. a, b miR-27a/b inhibit the delivery of damaged mitochondria to lysosome. 48 h post-transfection, HeLa cells stably expressing mtKeima were treated with CCCP or DMSO for 12 h. Cells were sequentially scanned using 440/10 nm (neutral) and 548/20 nm (acidic) excitation filters. Scale bars correspond to 100 μm. The signal intensity of the acidic Keima was divided by the intensity of the neutral mtKeima. Data were collected from 12 independent replicates and are shown as a percentage of control (n > 300 cells, two-way ANOVA). c, d miR-27a/b inhibit the degradation of COX2 upon mitochondrial damage. 48 h post-transfection, HeLa cells were treated with 10 μM CCCP or DMSO for 16 h. The degradation of damaged mitochondria was assessed by monitoring the levels of COX2, a mitochondrial inner membrane protein, by Western blot (n = 4, two-way ANOVA). Values are mean ± SEM (n.s. = non-significant, *p < 0.05, ***p < 0.001)
Induction of miR-27a/b under the chronic mitophagic flux condition
To determine if there is a regulatory interaction between miR-27a/b and mitophagy, we next examined the change in miR-27a/b levels upon mitochondrial damage at different time points. Interestingly, the levels of miR-27a/b were dramatically increased under the chronic mitophagic flux condition in HeLa cells (Fig. 9a, b). miR-27a and miR-27b are produced from miR-23a ~ 27a ~ 24–2 cluster at chromosome 19p13 and miR-23b ~ 27b ~ 24–1 cluster at chromosome 9q22, respectively (Additional file 8a). The mature miRNAs of miR-23a ~ 27a ~ 24–2 cluster are known to be derived from a single primary transcript [42]. The miRNAs of miR-23b ~ 27b ~ 24–1 cluster are transcribed independently but regulated by the same promoter [43]. Therefore, the expression of miRNAs in these clusters is expected to be regulated together. In the same manner with miR-27a/b, the levels of miR-23a/b were also increased under the chronic mitophagic flux condition (Additional file 8b, c). However, there was no change in the levels of several other miRNAs which are not included in these clusters (Additional file 8d). These data suggest that the change of miRNA expression under the chronic mitophagic flux is not a general nonspecific phenomenon affecting all miRNAs. Taken together, miR-27a/b are endogenous inhibitors of mitophagy and may function as a negative feedback loop under chronic mitophagic flux (Fig. 9c).
The levels of miR-27a/b are increased under chronic mitophagic flux condition. a, b HeLa cells were incubated with DMSO, 10 μM CCCP, or combination of 10 μM oligomycin and 4 μM antimycin as indicated. miR-27a/b levels were measured by qRT-PCR and normalized to corresponding U6 levels. Data are shown as fold changes relative to the DMSO controls. Values are mean ± SEM (t-test, **p < 0.01, ***p < 0.001). c A proposed role of miR-27a/b in mitophagy. Upon chronic mitophagic flux, the levels of miR-27a/b are increased and miR-27a/b function as a negative feedback loop to turn off excessive mitophagic flux. Upon the induction of miR-27a/b expression, the translation of PINK1 mRNA is inhibited by miR-27a/b. Consequently, the level of PINK1 protein in mitochondria decreases and thereby, autophagic degradation of the damaged mitochondria by lysosome is inhibited (Left panel). In contrast, PINK1 translation and lysosomal clearance of damaged mitochondria are increased when PINK1 inhibition mediated by miR-27a/b was disinhibited by the low levels of miR-27a/b (Right panel)
Mitochondria play critical roles in a variety of cellular functions, such as energy production, calcium homeostasis, and apoptosis [44]. Mitochondrial dysfunction has been suggested as a key factor in several diseases, including metabolic diseases and neurodegenerative diseases [44, 45]. Therefore, maintaining mitochondrial homeostasis is critical for cellular functions and health. In eukaryotic cells, mitochondrial homeostasis is maintained by mitochondrial quality control system which is a dynamic process coordinated by fission and fusion, mitophagy, transport, and biogenesis [46].
PINK1 plays critical roles in mitochondrial quality control, particularly in induction of mitophagy [39]. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, our understanding of PINK1 expression regulation at the transcriptional or translational levels, in particular, by miRNAs is still very limited. Here, we demonstrated that miR-27a/b negatively regulate human PINK1 through translational inhibition by directly targeting 3′UTR of its mRNA. Of note, our data suggest that PINK1 translation is critical for the accumulation of PINK1 protein upon mitochondrial damage. We further demonstrated that translational inhibition of PINK1 by miR-27a/b suppresses the autophagic clearance of damaged mitochondria by lysosome.
Our results demonstrated that endogenous PINK1 levels are actively regulated by the expression levels of miR-27a/b under the basal condition. Inhibition of endogenous miR-27a/b significantly increased the PINK1 protein levels under the basal condition. These data suggest that endogenous miR-27a/b critically contribute to maintaining the level of PINK1 protein lower than the threshold required for mitophagic induction and thereby, may protect cells from the untoward loss of mitochondria under the basal condition. Upon mitochondrial damage, mitophagic influx is regulated by miR-27a/b through their direct regulation of PINK1 expression, suggesting the roles of miR-27a/b as gate-keepers of mitophagic pathway. Interestingly, the expression of miR-27a/b is dramatically increased under the chronic mitophagic flux condition. Given the critical roles of mitochondria in cellular homeostasis and energy metabolism, the continuous loss of mitochondria will be toxic to the cells. Our data suggest that miR-27a/b may function as a negative feedback loop under the chronic mitophagic flux condition in order to protect cells from the depletion of mitochondria. Determining how the expression of miR-27a/b is regulated under the basal condition and mitochondrial stress condition will provide further insights into the regulatory mechanism of PINK1 and mitophagy.
miRNAs are getting more attention as critical regulators for many cellular processes. So far, only few miRNAs have been shown to regulate mitophagy. For example, miR-137 inhibits mitophagy by targeting FUNC1 and NIX [47]. miR-320a promotes mitophagy by targeting VDAC1 [48]. miR-351 and miR-125a are also known to regulate mitophagy [49]. These studies suggest that mitophagy is actively regulated by diverse miRNAs at multiple levels. In this study, we report that endogenous miR-27a/b play a critical role in PINK1 expression, thereby in mitophagy. Therefore, our study significantly contributes to understanding of the regulation mechanism of mitophagy, in particular, by miRNAs. Loss of PINK1 has been associated with mitochondrial dysfunction in Drosophila, mouse, and human, suggesting that the roles of PINK1 in mitochondrial quality control may be evolutionally conserved [50, 51]. Although the function of PINK1 may be conserved across evolution, our study suggests that miR-27a/b may exert a primate-specific regulation of PINK1 and mitophagy. miR-27a and miR-27b are produced from miR-23a ~ 27a ~ 24–2 and miR-23b ~ 27b ~ 24–2 cluster, respectively. These paralogous clusters also produce miR-23a, miR-23b, and miR-24. Interestingly, all of them are known to regulate autophagy-associated genes. For example, miR-23a, miR-23b, and miR-24 suppress AMBRA1 [52], ATG12 [53, 54], and ATG4A [55], respectively. Although the role of ATG4A in mitophagy is unclear, AMBRA1 and ATG12 positively regulate mitophagy [56–58]. Therefore, it is tempting to speculate that miRNAs in those clusters may coordinate their function to effectively regulate mitophagy.
PINK1 is expressed ubiquitously in neurons throughout the human brain [59, 60]. Several previous studies showed that miR-27a/b are expressed in human midbrain [34–36]. Because we demonstrated that endogenous miR-27a/b regulate PINK1 expression in human dopaminergic-like M17 cells, our study raises the possibility that miR-27a/b may regulate PINK1 expression in dopaminergic neurons of the human midbrain. Although the role of PINK1 in mitophagy in neurons is controversial [61], PINK1 also plays crucial roles in mitochondria trafficking, spine morphogenesis, and vulnerability to excitotoxicity in neurons [23]. Therefore, further studies are warranted to determine the roles of miR-27a/b in dopaminergic neurons of the human brain.
Recent miRNA profiling studies identified several miRNAs that are dysregulated in the affected regions of PD brains [35, 36, 62]. However, there are significant discrepancies between these studies, likely due to methodological differences as well as the heterogeneity of sample population, disease stages, and brain subregions examined. Moreover, relatively small sample sizes in those studies make it difficult to draw a firm conclusion whether expression of miR-27a/b and other miRNAs in miR-23a ~ 27a ~ 24–2 and miR-23b ~ 27b ~ 24–2 cluster are dysregulated in the brains of PD patients. Closer examination of miR-27a/b expression in different brain cell types at different stages of PD is needed to gain further insight into the putative roles of miR-27a/b in PD pathogenesis.
PINK1 is genetically associated with Parkinson's disease (PD) and plays critical roles in maintaining mitochondrial homeostasis. In this study, we identified a novel regulatory mechanism of PINK1 by miR-27a and miR-27b at the translational level. Our research highlights that protein translation step of PINK1 is critical for PINK1 accumulation upon mitochondrial damage. Consequently, the accumulation of PINK1 and mitophagic flux upon mitochondrial damage is strongly regulated by expression levels of miR-27a and miR-27b. Our results represent a significant advance in our understanding of translational control of PINK1 expression and mitophagy by miRNAs.
Human cervical HeLa and dopaminergic-like M17 cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, 11965084) or Opti-MEM I (Invitrogen, 31985070) with 10 % fetal bovine serum (FBS, Invitrogen, 16000044) and 1 % penicillin/streptomycin at 37 °C in a humidified 5 % CO2 incubator, respectively. Synthetic miR-27a/b and a negative control miR (Catalog # M-03-D) were from Insight Genomics. Anti-miR-27a/b (4101393–001) and anti-negative control (199006–001) were from Exiqon. AGO2 (E-004639-00-0005), DICER1 siRNA (M-003483-00-0005), and negative siRNA control (D-001206-14-05) were from Dharmacon. PINK1 siRNA (5′-GACGCTGTTCCTCGTTATGAA-3′) was from Qiagen.
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Total RNAs were extracted using TRIzol® Reagent (Invitrogen) and reverse transcribed with High Capacity cDNA Reverse Transcription kit (Applied Biosystems) for mRNAs or with Mir-X™ miRNA First-Strand Synthesis Kit (Clontech) for miRNAs except miR-27a/b according to the manufacturer's guides. Quantitative PCR was performed with Power SYBR Green PCR Master Mix, ABI 7500, and ABI 7900 (Applied Biosystems) using default thermal cycling program. PINK1 mRNA levels were measured with PINK1 forward primer: GGAGGAGTATCTGATAGGGCAG and reverse primer: AACCCGGTGCTCTTTGTCAC. miR-27a/b levels were measured as previously described [63]. Mature sequences and universal reverse primer were used for all other miRNAs. GAPDH and U6 were used as normalization controls for mRNA and miRNA, respectively.
AGO-specific RNA immunoprecipitation assay
RNA immunoprecipitation assay was carried out as previously described [64]. Briefly, HeLa cells were transfected with 50 nM of AGO2 siRNA or negative control. 48 h after transfection, AGO2-bound mRNAs were pulled down from cell lysates with 2A8 anti-AGO2 antibody. Then, RNAs were extracted using TRIzol® Reagent and PINK1 mRNA levels were measured by qRT-PCR.
Luciferase assay
The entire 3′UTR of human PINK1 mRNA (NM_032409, 1841–2660) was cloned into psiCHECKTM-2 vector at XhoI/NotI site downstream of Renilla luciferase. psiCHECKTM-2 vector also contains a constitutively expressed firefly luciferase gene that was used to normalize Renilla luciferase activity. Mutations in seed match sites were introduced using KOD-Plus-Mutagenesis Kit (TOYOBO). HeLa cells were plated at a density of 2.4 × 104 cells per well in a 96-well plate a day before transfection. Cells were transfected with 40 nM of miR-27a/b or negative control or with 150 nM of anti-miR-27a/b or anti-control along with 40 ng of luciferase reporter vector as indicated. 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega).
Mitochondrial fractionation
48 h post-transfection as indicated, cell were treated with 10 μM CCCP for 2 h and then, mitochondrial fraction was prepared as previously described [65].
High Content Imaging and immunofluorescence staining
For quantification of Parkin translocation, 40 nM miRNA was transfected to HeLa cells stably expressing GFP-Parkin in 384-well imaging plates. 48 h post-transfection, cells were treated with 10 μM CCCP for 2 h. Cells were then fixed in 4 % paraformaldehyde, stained with Hoechst 33342 (Invitrogen), and imaged on a BD Pathway 855 system (Becton Dickinson Biosciences). Parkin translocation was analyzed as previously described [40]. Analyzed were 4 independently transfected wells each with a total number of > 1000 cells per condition. After imaging on the BD pathway, plates were stained with mitochondrial marker, translocase of outer mitochondrial membrane 20 homolog (yeast) (Tom20), using anti-TOM20 antibody (Proteintech, 11802-1-AP). Confocal fluorescent images were taken with a 40 x Plan-Apochromat objective using a Zeiss AxioObserver equipped with an ApoTome Imaging System (Zeiss).
For the analysis of mitochondrial degradation, delivery of the damaged mitochondria to lysosome was visualized and quantified as previously described [41] with some modifications. Briefly, 40nM miRNA was transfected in HeLa cells stably expressing mitochondria-targeting Keima (mtKeima) in 384-well imaging plates. 48 h post-transfection, cells were treated with 4 μM CCCP for 12 h. Cells were imaged live after addition of Hoechst 33342 on the BD Pathway system. Acquisition was performed with a 2x2 montage (no gaps) after laser autofocus. 440/10 nm and 548/20 nm excitation filter were used for neutral and acidic Keima, respectively. Emission was filtered through a 595 nm longpass dichroic filter. 570 nm and 645/75 nm emission filters were used for neutral and acidic Keima, respectively. Raw images were processed using the build-in AttoVision V1.6 software. Regions of interest (ROIs) were defined as nucleus and cytoplasm using the build-in 'RING - 2 outputs' segmentation for the Hoechst channel after applying a shading algorithm. The signal intensity of the acidic Keima in the cytoplasm was divided by the intensity of the neutral mtKeima. Analyzed were 12 wells per condition with a minimum of 300 cells per well. PINK1 siRNA was used as a positive control for both assays.
Western blots were performed as previously described [66]. All membranes were blocked with 4 % non-fat dry milk in TBS/T (Tris buffered saline with 0.125 % Tween-20) except for pUbS65 which was blocked with 4 % bovine serum albumin (Jackson ImmunoResearch, 001-000-173) in TBS/T. Blots were probed in blocking buffer with anti-Dicer (Cell Signaling Technology, 5362), anti-PINK1 (Novus Biologicals, BC100-494), anti-SOD2 (Abcam, ab13533), anti-VDAC1 (Abcam, ab14734), anti-p38 (Cell Signaling Technology, 9212), anti-COX2 (Abcam, ab110258), or anti-GAPDH antibody (Santa Cruz, sc-25778). pUbS65-specific antibody were produced and characterized as previously described [19].
Statistical analyses were performed using two-tailed Student's t-test for the comparison of two groups. The comparison of multiple groups more than two were analyzed using one-way ANOVA or two-way ANOVA test depending on comparison variables with Tukey's pot-hoc analysis as indicated (GraphPad Prism 5).
AGO, Argonaute; CCCP, carbonyl cyanide m-chlorophenylhydrazone; COX2, cytochrome c oxidase 2; Dicer, dicer 1, ribonuclease type III; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LRRK2, Leucine-rich repeat kinase 2; miR-27a, microRNA-27a; miR-27b, microRNA-27b; miRNA, microRNA; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PD, Parkinson's disease; PINK1, PTEN-induced putative kinase 1; qRT-PCR, quantitative Real Time Polymerase Chain Reaction; SNCA, synuclein alpha; TOM20, translocase of outer mitochondrial membrane 20 homolog (yeast); UTR, untranslated region; VDAC1, voltage-dependent anion channel 1
Shulman JM, De Jager PL, Feany MB. Parkinson's disease: genetics and pathogenesis. Annu Rev Pathol. 2011;6:193–222.
Martin I, Dawson VL, Dawson TM. Recent advances in the genetics of Parkinson's disease. Annu Rev Genomics Hum Genet. 2011;12:301–25.
Exner N, Lutz AK, Haass C, Winklhofer KF. Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences. EMBO J. 2012;31(14):3038–62.
Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003;39(6):889–909.
Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet. 2006;38(5):515–7.
Kraytsberg Y, Kudryavtseva E, McKee AC, Geula C, Kowall NW, Khrapko K. Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat Genet. 2006;38(5):518–20.
Parker Jr WD, Parks JK, Swerdlow RH. Complex I deficiency in Parkinson's disease frontal cortex. Brain Res. 2008;1189:215–8.
Schapira AH, Cooper JM, Dexter D, Jenner P, Clark JB, Marsden CD. Mitochondrial complex I deficiency in Parkinson's disease. Lancet. 1989;1(8649):1269.
Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 1998;392(6676):605–8.
Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, et al. Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science. 2004;304(5674):1158–60.
Springer W, Kahle PJ. Regulation of PINK1-Parkin-mediated mitophagy. Autophagy. 2011;7(3):266–78.
Narendra DP, Jin SM, Tanaka A, Suen DF, Gautier CA, Shen J, et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 2010;8(1):e1000298.
Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, Gautier CA, et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J Cell Biol. 2010;189(2):211–21.
Zhang Y, Gao J, Chung KK, Huang H, Dawson VL, Dawson TM. Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proc Natl Acad Sci U S A. 2000;97(24):13354–9.
Shimura H, Hattori N, Kubo S, Mizuno Y, Asakawa S, Minoshima S, et al. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat Genet. 2000;25(3):302–5.
Kane LA, Lazarou M, Fogel AI, Li Y, Yamano K, Sarraf SA, et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol. 2014;205(2):143–53.
Kazlauskaite A, Kondapalli C, Gourlay R, Campbell DG, Ritorto MS, Hofmann K, et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J. 2014;460(1):127–39.
Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature. 2014;510(7503):162–6.
Fiesel FC, Ando M, Hudec R, Hill AR, Castanedes-Casey M, Caulfield TR, et al. (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation. EMBO Rep. 2015;16(9):1114–30.
Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW. The PINK1-PARKIN mitochondrial ubiquitylation pathway drives a program of OPTN/NDP52 recruitment and TBK1 activation to promote mitophagy. Mol Cell. 2015;60(1):7–20.
Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, Burman JL, et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature. 2015;524(7565):309–14.
Seirafi M, Kozlov G, Gehring K. Parkin structure and function. FEBS J. 2015;282(11):2076–88.
Deas E, Plun-Favreau H, Wood NW. PINK1 function in health and disease. EMBO Mol Med. 2009;1(3):152–65.
Chu CT. A pivotal role for PINK1 and autophagy in mitochondrial quality control: implications for Parkinson disease. Hum Mol Genet. 2010;19(R1):R28–37.
Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 2010;11(9):597–610.
Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–33.
Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet. 2011;12(2):99–110.
Cho HJ, Liu G, Jin SM, Parisiadou L, Xie C, Yu J, et al. MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein. Hum Mol Genet. 2013;22(3):608–20.
Xiong R, Wang Z, Zhao Z, Li H, Chen W, Zhang B, et al. MicroRNA-494 reduces DJ-1 expression and exacerbates neurodegeneration. Neurobiol Aging. 2014;35(3):705–14.
Doxakis E. Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. J Biol Chem. 2010;285(17):12726–34.
Junn E, Lee KW, Jeong BS, Chan TW, Im JY, Mouradian MM. Repression of alpha-synuclein expression and toxicity by microRNA-7. Proc Natl Acad Sci U S A. 2009;106(31):13052–7.
Meister G. Argonaute proteins: functional insights and emerging roles. Nat Rev Genet. 2013;14(7):447–59.
Kim J, Yoon H, Ramirez CM, Lee SM, Hoe HS, Fernandez-Hernando C, et al. MiR-106b impairs cholesterol efflux and increases Abeta levels by repressing ABCA1 expression. Exp Neurol. 2012;235(2):476–83.
Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007;129(7):1401–14.
Kim J, Inoue K, Ishii J, Vanti WB, Voronov SV, Murchison E, et al. A MicroRNA feedback circuit in midbrain dopamine neurons. Science. 2007;317(5842):1220–4.
Cardo LF, Coto E, Ribacoba R, Menendez M, Moris G, Suarez E, et al. MiRNA profile in the substantia nigra of Parkinson's disease and healthy subjects. J Mol Neurosci. 2014;54(4):830–6.
Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R. Fast and effective prediction of microRNA/target duplexes. RNA. 2004;10(10):1507–17.
Lennox KA, Behlke MA. A direct comparison of anti-microRNA oligonucleotide potency. Pharm Res. 2010;27(9):1788–99.
Pickrell AM, Youle RJ. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron. 2015;85(2):257–73.
Fiesel FC, Moussaud-Lamodiere EL, Ando M, Springer W. A specific subset of E2 ubiquitin-conjugating enzymes regulate Parkin activation and mitophagy differently. J Cell Sci. 2014;127(Pt 16):3488–504.
Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 2011;18(8):1042–52.
Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23(20):4051–60.
Sun F, Wang J, Pan Q, Yu Y, Zhang Y, Wan Y, et al. Characterization of function and regulation of miR-24-1 and miR-31. Biochem Biophys Res Commun. 2009;380(3):660–5.
Nunnari J, Suomalainen A. Mitochondria: in sickness and in health. Cell. 2012;148(6):1145–59.
Schon EA, Przedborski S. Mitochondria: the next (neurode)generation. Neuron. 2011;70(6):1033–53.
Baker MJ, Tatsuta T, Langer T. Quality control of mitochondrial proteostasis. Cold Spring Harb Perspect Biol. 2011;3:7.
Li W, Zhang X, Zhuang H, Chen HG, Chen Y, Tian W, et al. MicroRNA-137 is a novel hypoxia-responsive microRNA that inhibits mitophagy via regulation of two mitophagy receptors FUNDC1 and NIX. J Biol Chem. 2014;289(15):10691–701.
Li QQ, Zhang L, Wan HY, Liu M, Li X, Tang H. CREB1-driven expression of miR-320a promotes mitophagy by down-regulating VDAC1 expression during serum starvation in cervical cancer cells. Oncotarget. 2015;6(33):34924–40.
Barde I, Rauwel B, Marin-Florez RM, Corsinotti A, Laurenti E, Verp S, et al. A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy. Science. 2013;340(6130):350–3.
Narendra DP, Youle RJ. Targeting mitochondrial dysfunction: role for PINK1 and Parkin in mitochondrial quality control. Antioxid Redox Signal. 2011;14(10):1929–38.
Cookson MR. Parkinsonism due to mutations in PINK1, parkin, and DJ-1 and oxidative stress and mitochondrial pathways. Cold Spring Harb Perspect Med. 2012;2(9):a009415.
Zhang JA, Zhou BR, Xu Y, Chen X, Liu J, Gozali M, et al. MiR-23a-depressed autophagy is a participant in PUVA- and UVB-induced premature senescence. Oncotarget. 2016. doi:10.18632/oncotarget.9357.
An Y, Zhang Z, Shang Y, Jiang X, Dong J, Yu P, et al. miR-23b-3p regulates the chemoresistance of gastric cancer cells by targeting ATG12 and HMGB2. Cell Death Dis. 2015;6:e1766.
Wang P, Zhang J, Zhang L, Zhu Z, Fan J, Chen L, et al. MicroRNA 23b regulates autophagy associated with radioresistance of pancreatic cancer cells. Gastroenterology. 2013;145(5):1133–43. e12.
Pan B, Chen Y, Song H, Xu Y, Wang R, Chen L. Mir-24-3p downregulation contributes to VP16-DDP resistance in small-cell lung cancer by targeting ATG4A. Oncotarget. 2015;6(1):317–31.
Van Humbeeck C, Cornelissen T, Hofkens H, Mandemakers W, Gevaert K, De Strooper B, et al. Parkin interacts with Ambra1 to induce mitophagy. J Neurosci. 2011;31(28):10249–61.
Strappazzon F, Nazio F, Corrado M, Cianfanelli V, Romagnoli A, Fimia GM, et al. AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1. Cell Death Differ. 2015;22(3):419–32.
Radoshevich L, Murrow L, Chen N, Fernandez E, Roy S, Fung C, et al. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell. 2010;142(4):590–600.
Blackinton JG, Anvret A, Beilina A, Olson L, Cookson MR, Galter D. Expression of PINK1 mRNA in human and rodent brain and in Parkinson's disease. Brain Res. 2007;1184:10–6.
Gandhi S, Muqit MM, Stanyer L, Healy DG, Abou-Sleiman PM, Hargreaves I, et al. PINK1 protein in normal human brain and Parkinson's disease. Brain. 2006;129(Pt 7):1720–31.
Grenier K, McLelland GL, Fon EA. Parkin- and PINK1-dependent mitophagy in neurons: will the real pathway please stand up? Front Neurol. 2013;4:100.
Minones-Moyano E, Porta S, Escaramis G, Rabionet R, Iraola S, Kagerbauer B, et al. MicroRNA profiling of Parkinson's disease brains identifies early downregulation of miR-34b/c which modulate mitochondrial function. Hum Mol Genet. 2011;20(15):3067–78.
Zhu Y, Zhang X, Ding X, Wang H, Chen X, Zhao H, et al. miR-27 inhibits adipocyte differentiation via suppressing CREB expression. Acta Biochim Biophys Sin (Shanghai). 2014;46(7):590–6.
Wang WX, Wilfred BR, Hu Y, Stromberg AJ, Nelson PT. Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes. RNA. 2010;16(2):394–404.
Geisler S, Holmstrom KM, Treis A, Skujat D, Weber SS, Fiesel FC, et al. The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations. Autophagy. 2010;6(7):871–8.
Kim J, Yoon H, Horie T, Burchett JM, Restivo JL, Rotllan N, et al. microRNA-33 Regulates ApoE Lipidation and Amyloid-beta Metabolism in the Brain. J Neurosci. 2015;35(44):14717–26.
Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res. 2008;36(Database issue):D149–53.
Dweep H, Sticht C, Pandey P, Gretz N. miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes. J Biomed Inform. 2011;44(5):839–47.
Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15–20.
We are grateful to Atsushi Miyawaki (Brain Science Institute, RIKEN, Japan) for sharing the mitochondria-targeting Keima construct.
This work was supported, in part, by Mayo Clinic Foundation (W.S), Marriott Family Foundation (W.S), Gerstner Family Career Development Award (W.S), GHR Foundation (J.K.), Mayo Clinic Center for Individualized Medicine (J.K. and W.S), Center for Regenerative Medicine (W.S.), the Center for Biomedical Discovery (W.S.), Neuroscience Focused Research Team Award (W.S.), the Michael J. Fox Foundation for Parkinson's Research (W.S.), the Foundation for Mitochondrial Medicine (W.S.), NIH grants AG016574 (J.K.), AG005681 (J.K), AG028383 (P.T.N), NS085830 (P.T.N) and NS085070 (W.S). F.C.F. is the recipient of a fellowship from the American Parkinson Disease Foundation (APDA).
The datasets supporting the conclusions of this article are included within the article and its additional files.
JK (Jaekwang Kim) and J.K. (Jungsu Kim) designed research; JK (Jaekwang Kim), FCF, PTN, WS, and JK (Jungsu Kim) wrote the paper; JK (Jaekwang Kim), FCF, KCB, RH, WW, CK, PTN, WS, and JK (Jungsu Kim) performed research; JK (Jaekwang Kim), WW, and FCF analyzed data. All authors read and approved the final manuscript.
Current address: CK, Gladstone Institute of Neurological Disease, University of California, San Francisco 94158, USA, chaeyoung.kim@gladstone.ucsf.edu; KCB, School of Graduate Studies, Health Sciences Center New Orleans, Louisiana State University, krystalbelmonte@gmail.com.
Competing interest
Ethical approval and consent to participate
Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Rd S, Jacksonville, FL, 32224, USA
Jaekwang Kim, Fabienne C. Fiesel, Krystal C. Belmonte, Roman Hudec, Chaeyoung Kim, Wolfdieter Springer & Jungsu Kim
Department of Pathology, University of Kentucky, Lexington, KY, 40536, USA
Wang-Xia Wang & Peter T. Nelson
Neurobiology of Disease Program, Mayo Graduate School, Jacksonville, FL, 32224, USA
Wolfdieter Springer & Jungsu Kim
Jaekwang Kim
Fabienne C. Fiesel
Krystal C. Belmonte
Roman Hudec
Wang-Xia Wang
Chaeyoung Kim
Peter T. Nelson
Wolfdieter Springer
Jungsu Kim
Corresponding authors
Correspondence to Jaekwang Kim or Jungsu Kim.
Knock-down of DICER1 decreased miRNA levels. HeLa cells were transfected with 50 nM of DICER1 siRNA (DICER1 KD) or negative control (Ctl). 48 h post-transfection, representative miRNA levels were analyzed by qRT-PCR and were quantified as a percentage of control (n = 2). Values are mean ± SEM. (PDF 18 kb)
Computational prediction of miRNA candidates for human PINK1. a Computation prediction of miRNAs expressed in human midbrain with putative binding sites in the 3′UTR of human PINK1 mRNA. We first searched miRNAs that have putative binding sites in the 3′UTR of human PINK1 mRNA by utilizing several miRNA-target prediction algorithms, such as miRanda [67], miRWalk [68], RNAhybrid [37], and Targetscan [69]. Among 49 miRNAs commonly predicted by different algorithms, 7 miRNAs were known to be expressed in human midbrain [34]. miR-27a/b are predicted to have 2 putative binding sites in the 3′UTR of human PINK1 mRNA, while all other miRNAs are predicted to have 1 putative binding site. b Computational binding prediction of miR-27a/b and their binding sites in the 3′UTR of human PINK1 mRNA. The binding free energies were determined by the RNAhybrid algorithm. (PDF 68 kb)
a Comparison of 3′UTR sequences of PINK1 mRNAs from different species. Multiple alignment was performed using ClustalW program with 3′UTR sequences of PINK1 mRNAs from human (NM_032409), chimpanzee (XM_001164912), monkey (XM_001096957), rat (NM_001106694), and Mouse (NM_026880). Seed match sites for miR-27a/b are highlighted in red. b A phylogenetic tree of 3′UTR sequences of PINK1 mRNA from different species. The neighbor-jointing tree for the full-length 3′UTR sequences of PINK1 mRNAs was generated with ClustalW program. (PDF 27 kb)
The levels of miR-27a (a) and miR-27b (b) in the luciferase assay. HeLa cells were transfected with miR-27a/b or negative control (Ctl) along with the reporter constructs as indicated in the X axis. 48 h post-transfection, miR-27a/b levels were measured by qRT-PCR. Each level was normalized to the corresponding U6 level. Data are shown as a fold change relative to the control miR (n = 5, one-way ANOVA). Values are mean ± SEM (n.s. = non-significant). (PDF 29 kb)
The levels of miR-27a/b after overexpression (a) and inhibition (b) of miR-27a/b. HeLa cells were transfected with 40 nM of negative control (Ctl) or miR-27a/b (A) or with 150 nM of anti-control (anti-Ctl) or anti-miR-27a/b (B). 48 h post-transfection, miR-27a/b levels were determined by qRT-PCR. Each level was normalized to the corresponding U6 level. Data are shown as a fold change relative to control (n = 4). Values are mean ± SEM (n.s. = non-significant, ***p < 0.001, one-way ANOVA (A) or t-test (B)). (PDF 28 kb)
A combination of miR-27a and miR-27b does not further inhibit PINK1 expression compared to each miRNA alone. HeLa cells were transfected with 80 nM negative control (Ctl), 40 nM Ctl with 40 nM miR-27a (miR-27a), 40 nM Ctl with 40 nM miR-27b (miR-27b), or 40 nM miR-27a with 40 nM miR-27b (miR-27a/b). 48 h post-transfection, PINK1 levels were measured by Western blot and normalized to corresponding GAPDH levels. Data are shown as a percentage of control (n = 4, one-way ANOVA). Values are mean ± SEM (***p < 0.001). (PDF 42 kb)
Knock-down of PINK1 prevents the delivery of damaged mitochondria to lysosome. 48 h post-transfection with negative control (si-Ctl) or PINK1 siRNA, HeLa cells stably expressing mtKeima were treated with 4 μM CCCP or DMSO for 12 h. Cells were sequentially scanned using 440/10 nm (neutral) and 548/20 nm (acidic) excitation filters. Scale bars correspond to 100 μm. The signal intensity of the acidic Keima was divided by the intensity of the neutral mtKeima (a). Data were collected from 12 independent replicates and are shown as a percentage of control (n > 300 cells, two-way ANOVA) (b). Values are mean ± SEM (n.s. = non-significant, ***p < 0.001). (PDF 291 kb)
a Schematic diagram of miR-23a ~ 27a ~ 24–2 and miR-23b ~ 27b ~ 24–1 clusters. b, c The levels of miR-23a/b upon mitochondrial damages. HeLa cells were incubated with DMSO, 10 μM CCCP, or combination of 10 μM oligomycin and 4 μM antimycin as indicated. miR-23a/b levels were measured by qRT-PCR and normalized to corresponding U6 levels. Data are shown as a fold change relative to DMSO control. d The levels of miR-103, miR-221, and miR-7a upon mitochondrial damages were measured by qRT-PCR and normalized to corresponding U6 levels. Values are mean ± SEM (t-test, ***p < 0.001). (PDF 41 kb)
Kim, J., Fiesel, F.C., Belmonte, K.C. et al. miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1). Mol Neurodegeneration 11, 55 (2016). https://doi.org/10.1186/s13024-016-0121-4
Received: 12 February 2016
miR-27a
miR-27b


The relentless investment in know-how and search for future proof innovative solutions helped us gain the long-term trust of regional and global partners and Frigotehnica took on the challenge to provide the same high-quality solutions in foreign projects since 2004.With the increase in demand for external support and installations Frigotehnica grew into a powerful regional player expanding with permanent offices first into Bulgaria and Ukraine and expecting to create in the near future a permanent footprint also in other countries of the region.
From 2015 until now we continue to expand our business in the region by signing contracts in Bulgaria and Ukraine.
We sign over 50 projects on the international market for one of the biggest logistics centers in Czech Republic and Slovakia.
The first CO2 subcritical project is in Hungary.
Czech Republic, Slovakia, Bulgaria and Ukraine.
We sign over 50 projects on the international market for one of the biggest logistics center in Czech Republic, after we continue in Slovakia, Bulgaria and Ukraine.
We set up the first branch in Bulgaria and Ukraine.


Almost all commodities or products these days are sold in various units and are created by different manufacturing companies. This gives people more options for every product. In today's world where most people have different preferences. Having different choices gives you a chance to know what could be and what other benefits are there if you choose a different choice. Hence, more options can be more beneficial for every buyer.
This can also be observed when you're buying any type of supplements. Some are thinking of using drugs that will help them improve brain functions and to prevent the deterioration of the performance of your entire body. In this aspect, having varied choices can be very helpful. It allows you to explore your options properly and consider what is needed. But there can be a downside for this. What can this be? It's the confusion you'll feel when faced with different choices. And since it's something that highly affects the overall body function, the person feels even more pressured to choose because of this.
And to effectively avoid confusion, you'll need something that can be used as an important reference of the current options available.
What do you NEED? You'll need to be specific about the different needs you have. Every supplement formula targets a certain brain function. When there's a certain part of the brain you wish to improve, you have to focus on that area and use products that'll help you achieve the improvements. This way, you'll be sure of the item you'll need in the future.
Brand for it. The manufacturing company can be a small thing for others but this can be a very big reference when you are considering the choices you have. There are brands which are highly famous because of the effects they consistently provide for their clients. These are the reputations of the companies you should rely on. Once you filter them properly, the list becomes shorter and more suitable for your needs.
How much are you willing to spend for it? Quality and cost must be properly balanced. If this isn't achieved, this can become the cause of difficulties for you. Even if you say that you're willing to invest when it comes to health and wellness, there will always be limits when it comes to how much you're spending for it.
Method for purchasing. These days, there are different methods for purchasing. The traditional option is still what many prefer. But there are many who are shifting to online purchases because of its convenience and the different benefits that it provides. It's important to consider these options.
Whether you're thinking of an actual purchase or considering an online transaction, it's important to start with learning where to go and which brand to use. It's important to know about the most effective brand and things that are needed. Having something you can trust when you're choosing the supplement will help with your needs. This can benefit your brain functions and stabilize a lot of bodily processes. Hence, it's highly necessary to consider the different needs you may have in the future.
If you browse this site, you can be able to know about the different options and see one of the most reputable brands and the numerous products under it which can help you.
NEXT POST Next post: How Living in Canada is Worth ?


1520 Sweden: Christian II becomes King: the murder of 82 notable people in Stockholm, known as the Stockholm Bloodbath, earns him the nickname "Christian the Tyrant."
1532 Wallachia: Vlad the Drowned is so named after he gets drunk and rides his horse into the Dambovita River where he drowns.
1590 Rome: Cardinal Giovanni Battista Castagna is elected pope as Urban II and dies of malaria after 13 days, making him the shortest reigning pope in history.
We're waiting like everyone on the east coast to see if the hurricane turns into the "perfect superstorm" that the forecasters are predicting. Fritz has pulled all the potted plants into the house for the winter, but the autumn gardens are still very lovely.
A bank of chrysanthemums with our abstract cityscape behind. Some of those "skyscrapers" will probably go over in the wind so we may have to do some urban renewal after the storm passes.
The flower plumes of one of our stands of ornamental grass sparkle as if they're made of crystal beads when the sun is behind them. I love them like this. This group is right outside the living room windows.
A Kusa Dogwood whose various reds, oranges and yellows glow as if lit from within when the setting sun backlights it at just the right angle.
Starr, on rug and in the sunlight of a perfect late October day.
A new structure rising on the concrete patio outside the Center. The idea was mine, that we should have our own maple sugaring shack where the boiler/evaporator unit could be housed permanently. This would eliminate having to put an awkward, heavy piece of equipment with a fair number of sharp edges into some kind of vehicle and then get them into the barn when sugaring is over in the spring, only to have to reverse the process late in the next winter.
I drew up a plan for a low wall around three sides of the boiler made of concrete block and the idea was that Fritz's nephew, who is in residence in the old house (background), would build the open-sided shed. He planned a much larger structure than we had envisioned but it's turning out to be very attractive and his mortise and tenon joints in the post and beam frame are true things of beauty. The same red metal roofing that's on the house will be on the shed.
Several cords of firewood are stacked under the center's balcony. I spent the day carrying the last of the wood from the dead trees we cut down in September down to the pile, so that's done for the season and we're ready for the winter!
from the media, this storm is set to wipe out all of the northeast, wash away all you wicked Godless democrats for your wicked ways.
The "shack" is truly beautiful. I hope you'll continue to post progress photos and, of course, the finished work. And now I have woodpile envy. My supply is not as large as I'd like this year. I'm hoping for a mild season!
Good luck with the storm. I hope the new shack survives the winds. From your description of the joints, Will, it sounds as though it should.
It does sound as if the storm may not have been overrated. Hope it is, but if not, trust you hunker down safely.
Hope all the loveliness in the photos here endures the storm. High winds here in North Georgia toppled an enormous oak next door in the neighborhood yesterday. I cannot imagine the reality of being in the path of an authentic hurricane. Best to you, take care.
I'm curious about the "skyscraper" installation. Can you elaborate about it in a post? Or have you already and I missed it?


text:
Biggest UK Online Casino Winners
Traditional vs. modern slot machines: Which were easier to beat?
January 2021: Top Slots To Try Out
Online Casino Chan Review - A Complete Details
Hiring Expert Locksmith Services in Orlando
The History of Online Slots & Their Evolution into Mainstream Gaming
Understanding Sports Betting Odds
Pelosi: Trump Could Be 'Accessory to Murder'
Empty shelves due to Covid-19 not Brexit - Brandon Lewis
Loud explosion rocks central Madrid, rescue teams to site
Quake on Indonesia's Sulawesi Island leaves thousands homeless as recovery begins
Snow could reduce visibility for Sault and area
MORE NEWS THIS CATEGORIES
Cayetano says Kuwait filed kidnapping raps vs Filipino diplomats
The foreign affairs secretary said only a senator and a former congressman wanted him to quit. The Foreign Affairs Department said it can not fully disclose d...
'Constitutional carry' bill advances through Oklahoma Senate in late-night session
Dahm claims that the announcement would have no result on places where carrying a weapon would be illegal, like colleges, basic schools and government arrangeme...
Greitens impeachment to be considered in special session, Missouri lawmakers say
A House report released Wednesday also presented evidence that the governor filed false information with the Missouri Ethics Commission. It comes as Greitens ...
Sheriff's Office warns residents to beware of phone scam
Calls have been made to citizens from someone claiming to represent the county sheriff's office. The message initially appears to be legitimate and asks that ...
Black bear spotted on West Side removed by DNR
The Grand Rapids Police Department said that a black bear was spotted in a tree on Dayton Street between Straight and Deloney avenues on the West Side. Wild bea...
13 miners trapped after Masakhane mine rockfall
The firm's James Wellsted said: "We had a seismic event about three kilometres underground at our Driefontein mine and it resulted in a fall of ground in one of...
Congress government turned Bengaluru into sin valley: Modi
PM Modi charged the ruling Congress with destroying Bengaluru . "The BJP-RSS (Rashtriya Swayamsevak Sangh) duo doesn't care about Karnataka's aspiration...
Farenthold: 'I Shouldn't Be Asked To Pay' For Special Election
In the letter, which was posted to his campaign website, Farenthold countered Abbott's request for him to pay the state back for allegedly using $84,000 in ta...
Man Accused of Churchville Double Murder Found Dead
By that time, there was an all-out police search for Mooney as a person of interest in the stabbing and shooting deaths of Tyler Christopher Roy, 28, and his wi...
SANA: Over 200 terrorists evacuated from Yarmouk camp in Damascus
The pictures were taken in Yarmouk, an area controlled by Isis and not far from Damascus, and is believed to be in retaliation to airstrikes by Syrian and Rus...
12-year-old Arrested for Bringing Replica Gun to School
The replica gun a Montgomery County middle school student allegedly brought to school. Montgomery County police said the 12-year-old boy brought the replica g...
Fallen Girard police officer honored during ceremony
He was trying to serve a warrant on a suspect, who then ran to his father's house. The eight-year veteran of the Elyria Police Department was on his way to pi...
Institutional Investors Are Dumping MTS Systems Corp (NASDAQ:MTSC)
MTSC stock traded up $0.30 during trading on Wednesday, hitting $51.90. 27,157 shares of the company traded hands, compared to its average volume of 72,142. 2,...
Waiting for 'green light' to leave hospital — George HW Bush
Houston Methodist Hospital has named the atrium of a new building after former President George H.W. Bush and his late wife, Barbara. Though he described the ...
Malaysia's ex-PM Mahathir under investigation for fake news
On Wednesday Malaysian police said Mahathir was being investigated under the fake news legislation, which was recently rushed through parliament by the prime ...
Fire breaks out at chemical plant in Louisiana, prompting evacuations
Officials say that if you have been evacuated, please wait for notifications to return to your residence. Louisiana State Police have reduced the evacuation r...
China seeks soybean boost for trade war advantage
According to Beijing's proposed retaliation plan, United States soybeans could face 25 percent tariffs when exported to China. Chinese authorities have ordere...
Adoption Special-Cinco De Meow in the City
Hours of Operation for Columbia Animal Services includeMonday - Friday 8:30 a.m. - 5:00 p.m. and Saturday 9:00 a.m. - 4:00 p.m. Drivers should be prepared to pr...
Gotcha - RCMP Dog Enzo Nabs Three Suspects
The 18-year-old woman was placed under arrest for Possession of Stolen Property. They also learned three other people had been in the vehicle, and began searchi...
Miami police officer recorded kicking suspect in head
A Florida police officer has been relieved of duty after a video surfaced showing him kicking a handcuffed man in the head after the man was already on the gr...
Horse sues ex-owner for $100000 after neglect
The suit claims that OR law and legal precedent has established that a horse is a 'sentient being, ' and can be 'the victim of the crime-an animal entitled to s...
Israeli Satellite Images Show Renewed Activity at Iran's Fordo Enrichment Facility
According to the newspaper, the Israeli company ImageSat released images Twitter, confirming that the entrance gate on the territory of an important energy faci...
California cops hunt paroled sex offender after RV chase
Houk is a registered sex offender who was on parole following a conviction in OR for felony sodomy, records indicated. The Los Angeles County Sheriff's Depa...
Police responding to active shooter in Mill Valley
Schneider said police who initially arrived on the scene Thursday afternoon carried two shooting victims from the scene and they were taken to a hospital. "At t...
Body found along I-4 in Polk County after hit-and-run, FHP says
The man whose body was found on the side of Interstate 4 on Thursday morning is thought to have been killed in a hit-and-run crash, troopers said. The victim ...
59 year old arrested following incident at polling station
Voters are going to the polls for a Westminster by-election in the West Tyrone constituency. A spokesman for Northern Ireland's Electoral Office (EONI) sa...
Trump May 'Get Involved' To Stop Mueller Probe, He Warns
You can challenge the subpoena in court, but you're going to lose that challenge and the only thing you will have done is delayed it and kept the investigation...
Trump to Hold Rally in South Bend
South Bend residents, clear your schedules! See what the Michiana community is saying on Facebook . The South Bend trip will be President Trump's second ...
Backup cameras now required in new cars in the US
When a driver shifts a vehicle into reverse, the RVS (rearview video system) shows-either in the dashboard or in a small display in the rearview mirror-an image...
Oil falls as United States production hits 2018 high
US crude stocks rose last week as refineries cut output, while gasoline stocks increased and distillate inventories fell, the Energy Information Administration ...
Back 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770 1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944 1945 1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091 2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146 2147 2148 2149 2150 2151 2152 2153 2154 2155 2156 2157 2158 2159 2160 2161 2162 2163 2164 2165 2166 2167 2168 2169 2170 2171 2172 2173 2174 2175 2176 2177 2178 2179 2180 2181 2182 2183 2184 2185 2186 2187 2188 2189 2190 2191 2192 2193 2194 2195 2196 2197 2198 2199 2200 2201 2202 2203 2204 2205 2206 2207 2208 2209 2210 2211 2212 2213 2214 2215 2216 2217 2218 2219 2220 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 2242 2243 2244 2245 2246 2247 2248 2249 2250 2251 2252 2253 2254 2255 2256 2257 2258 2259 2260 2261 2262 2263 2264 2265 2266 2267 2268 2269 2270 2271 2272 2273 2274 2275 2276 2277 2278 2279 2280 2281 2282 2283 2284 2285 2286 2287 2288 2289 2290 2291 2292 2293 2294 2295 2296 2297 2298 2299 2300 2301 2302 2303 2304 2305 2306 2307 2308 2309 2310 2311 2312 2313 2314 2315 2316 2317 2318 2319 2320 2321 2322 2323 2324 2325 2326 2327 2328 2329 2330 2331 2332 2333 2334 2335 2336 2337 2338 2339 2340 2341 2342 2343 2344 2345 2346 2347 2348 2349 2350 2351 2352 2353 2354 2355 2356 2357 2358 2359 2360 2361 2362 2363 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374 2375 2376 2377 2378 2379 2380 2381 2382 2383 2384 2385 2386 2387 2388 2389 2390 2391 2392 2393 2394 2395 2396 2397 2398 2399 2400 2401 2402 2403 2404 2405 2406 2407 2408 2409 2410 2411 2412 2413 2414 2415 2416 2417 2418 2419 2420 2421 2422 2423 2424 2425 Forward
Egypt, UAE Resume First Qatar Flights since 2017
Thai woman gets 43-year sentence for insulting king
Italian PM wins crucial vote in Senate with very thin margin
Germany to extend lockdown to mid-February on fears of COVID variants
Fox News Digital Laying Off Staffers
Navalny's Allies Plan 'Large Rallies' In Russia Following His Arrest
Utah National Guard troops deployed to D.C. for inauguration
Australia unlikely to fully open its borders in 2021
Russia's Gazprom says Nord Stream 2 at risk after U.S. sanctions
Pompeo: Chinese Genocide Against Muslims 'Crime of the Century'
The related cryptocurrency, Filecoin, raised $257 million for IPFS c...
The 2021 Taycan is expected to arrive at USA dealerships starting ear...
The Chromebook convertible models have been announced to "meet the nee...
In the announcement post, Blizzard adds that there are some mechanics ...
Qualcomm also threw shade at competitors by claiming its chip is creat...
Well, the DC Extended Universe is still looking for a way to execute i...
The host asked if the rumours were true or not, the beauty is finally ...
Here is the synopsis: "A couple gathering their family together for a ...
Dave Grohl vividly recalled visiting Lemmy Kilmister at the Motorh...
Other artists read names of Black victims including George Floyd , W...
Spot gold rose 1.5% to $1,867.06 per ounce by 11:58 a.m. EST (1658 GMT...
The agency noted that methane emissions have decreased by 10% in the p...
European Council President Charles Michel arrives for a video conferen...
The fishermen are in turmoil after facing a series of problems exporti...
In another survey reported by Reuters, this time from Deutsche Ban...
© 2021 clicklancashire.com, Click Lancashire

redpajama_set_name: RedPajamaCommonCrawl


This sweet little Waxwing stands at 15.5cm tall. He is made entirely by hand, by Abigail Brown, from both new and reused materials, carefully selected the replicate the colours and markings of the Waxwing. He is both machine and hand embroidered, with immense detailing. A truly beautiful little piece to delight any home or display space. Your Waxwing will be made to order and can take some time, please enquire before purchasing. As a handmade piece no two will ever be the same so you must expect variations from the one shown here.


Here's how to make the most out of your daily steps!
Achievement pays out $10 via check or Paypal when you've accumulated 10,000 points! This takes some time, but this app takes zero effort. Literally just set it up and forget it until you can cash in on your points!
If you have a Fitbit, all you have to do is link Achievement to the app! You can also link it to the "Health" app on an iPhone to track your steps. There are a number of other apps you can link Achievement to including Map My Run, Google Fit, Microsoft Health, My Fitness Pal, and many others! Achievement will reward you for keeping track of your food and water intake as well as your weight! You can be rewarded for other exercises as well. If you don't want to go the extra mile of logging all these different categories, you can just set it to the appropriate step tracker for your device and cash in on all that walking!
Sign up for Achievement here!
Sweatcoin is a little trickier than Achievement. You only get credit for outdoor steps logged with your phone. So not only do you have to be outside, you have to have your phone on you as well. Sweatcoin uses an algorithm to check how far apart your steps are to keep people from cheating—completely understandable, but makes the app slightly less appealing. Especially, for those of us that like to do our walking on a treadmill.
Sweatcoin requires you to either have the app open in the background or to set it up with the built in step tracker on your phone (i.e. the Health App on iPhone). The latter may result in some missed steps, though. The app will convert your steps into Sweatcoins that you can then redeem.
Approximately 2,000 steps is equal to 1 Sweatcoin. There are currently four "subscription" levels within the app. The "Mover" subscription is free and allows you to earn up to five Sweatcoins a day. The next level up allows you to earn up to 10 Sweatcoins a day, but will cost you just under five Sweatcoins a month. You can subscribe to whichever level suits your earning the best!
Rewards work a little differently, too. You earn "Sweatcoins" that you can cash in for offers. These aren't always cash, but rather products and discounts. If you really want a cash payout, you'll have to save up or get some of your friends to join! The more invites you get, the more cash payouts become available. It's an interesting system. You can find some cool new companies to try for free!
Both apps are completely free! If you walk a lot in your day to day life, they're worth checking out and earning some easy cash and rewards! Happy earning!


Get the App. Download the free InterBank App to have banking information at your fingertips. Check balances, make transfers or check deposits, find InterBank locations or ATMs, all on your smartphone.
Get a weekly dose of forex. Inthe Central Bank of Nigeria introduced what was supposed nigeria be an autonomous foreign forex market. The intention was interbank make it easier to interbank foreign exchange to end-users through authorised dealers and at a rate set by the market.
3/13/2019 · The Bank of Ghana (BoG) in attempt to halt the cedi from further sharp decline fortnight ago announced the new Ghana Interbank Forex Market Conduct directives, which among other things, spell out who is eligible to trade in forex in the country, how records should be kept and reported to the Bank of Ghana, and some general rules on the trading of forex.
For example, at the interbank market, the CBN exchange rate of dollars is at a dynamic equilibrium of 305.5 Naira giving liberty to the banks to sell at higher price rate. One such bank that we would discuss it's dollar to naira exchange rate is that of GTBank Mastercard exchange rate Today Nigeria.
© Interbank forex rate in ghana Binary Option | Interbank forex rate in ghana Best binary options.