Abstract
Dysfunction of growth factor (GF) activities contributes to the decline and death of neurons during aging and in neurodegenerative diseases. In addition, neurons become more resistant to GF signaling with age. Micro (mi)RNAs are posttranscriptional regulators of gene expression that may be crucial to age- and disease-related changes in GF functions. MiR-126 is involved in regulating insulin/IGF-1/phosphatidylinositol-3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling, and we recently demonstrated a functional role of miR-126 in dopamine neuronal cell survival in models of Parkinson’s disease (PD)-associated toxicity. Here, we show that elevated levels of miR-126 increase neuronal vulnerability to ubiquitous toxicity mediated by staurosporine (STS) or Alzheimer’s disease (AD)-associated amyloid beta 1–42 peptides (Aβ1–42). The neuroprotective factors IGF-1, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and soluble amyloid precursor protein α (sAPPα) could diminish but not abrogate the toxic effects of miR-126. In miR-126 overexpressing neurons derived from Tg6799 familial AD model mice, we observed an increase in Aβ1–42 toxicity, but surprisingly, both Aβ1–42 and miR-126 promoted neurite sprouting. Pathway analysis revealed that miR-126 overexpression downregulated elements in the GF/PI3K/AKT and ERK signaling cascades, including AKT, GSK-3β, ERK, their phosphorylation, and the miR-126 targets IRS-1 and PIK3R2. Finally, inhibition of miR-126 was neuroprotective against both STS and Aβ1–42 toxicity. Our data provide evidence for a novel mechanism of regulating GF/PI3K signaling in neurons by miR-126 and suggest that miR-126 may be an important mechanistic link between metabolic dysfunction and neurotoxicity in general, during aging, and in the pathogenesis of specific neurological disorders, including PD and AD.
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Introduction
Growth factor (GF) signaling pathways are essential for the function and survival of neurons, and their dysfunction has been implicated in the decline or death of neurons during aging and in neurodegeneration. In particular, insulin/IGF-1 signaling pathways have been associated with age-related neuronal dysfunction and neurodegenerative diseases, such as Parkinson’s (PD) and Alzheimer’s disease (AD) [1–8]. The detailed mechanisms of GF dysfunctions in aging and neurodegeneration, however, are still not well understood. One current hypothesis is that neurons become more resistant to GF actions over time [4–6, 8, 9].
Micro (mi)RNAs regulate gene expression at the posttranscriptional level [10]. They are involved in all aspects of cell functions, including key signaling pathways that are important in the maintenance of cellular homeostasis and response to stress. There is evidence that miRNAs are involved in neuronal aging and in the pathogenesis of neurodegenerative disorders, but their precise relationships with GF activities are poorly understood [11–18]. In nonneuronal cells, IGF-1/phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling is, in part, modulated by miR-126 (reviewed in [14, 19]), and in hepatocytes, upregulation of this miRNA has been associated with insulin resistance [20]. miR-126 has also been described in the neuronal context [21–27], and we have recently shown that miR-126 was upregulated in dopamine (DA) neurons in postmortem PD patients’ brains and in pyramidal cortical neurons from schizophrenia patients [14, 28, 29]. Moreover, our recent data showed that elevated levels of miR-126 in the DA neuronal context is neurotoxic to 6-hydroxydopamine (6-OHDA) by impairing IGF/PI3K/AKT and ERK signaling, while its inhibition is neuroprotective [28].
Because of the critical roles of GF-activated PI3K/AKT and ERK signaling in neuronal function and age- or disease-associated dysfunction, we hypothesized that miR-126 might play a general role in regulating or deregulating the effects of a variety of GFs in neurons, including nerve growth factor (NGF) whose diminished trophic effects on cholinergic neurons has been linked to cognitive decline in aging and AD [30–33]; soluble amyloid precursor protein α (sAPPα, one of the cleaved products of amyloid precursor protein (APP)) that has neurotrophic and neuroprotective properties [34, 35], acts synergistically with NGF and IGF-1, and can reverse the toxic effects of amyloid beta (Aβ) [36, 34], another product of APP cleavage which may be the primary toxic agent in AD pathogenesis [37, 35]; and brain-derived neurotrophic factor (BDNF) which is involved in neuroplasticity and protection and has been associated with aging and a variety of neuropsychiatric and neurodegenerative disorders [38, 39].
Here, we show that elevated levels of miR-126 in cortical and hippocampal neurons are neurotoxic and enhance the effects of ubiquitous toxicity mediated by staurosporine (STS), a general kinase inhibitor [40, 41], and cell-specific toxicity due to Aβ1–42 peptides, which directly interact with GF/PI3K signaling pathways [8, 34, 42, 43]. Neurotoxicity could be diminished, but not abrogated, by IGF-1, NGF, BDNF, and sAPPα, and inhibition of miR-126 was neuroprotective. In neurons derived from Tg6799 mice, which is a model of familial AD (FAD) [44], we observed an increase in miR-126 expression and Aβ1–42 toxicity, but surprisingly, and in contrast to littermate controls, both Aβ1–42 and miR-126 promoted neurite sprouting. On the mechanistic level, overexpression of miR-126 caused a downregulation of factors in the PI3K and ERK signaling cascades. Our data provide evidence for a novel mechanism of regulating GF/PI3K signaling in neurons by miR-126 and suggest a functional role of this miRNA, broadly, in aging neurons, and the pathogenesis of neurodegenerative diseases.
Material and Methods
Lentivirus Vectors and Cell Transduction
The third generation lentivirus system was kindly provided by Drs. D. Trono and R. Zufferey, University of Geneva, Switzerland [45, 46]. For neuron-specific expression of rno-miR-126, the Synapsin promoter was subcloned from pHIV7/Syn-EGFP (kindly provided by Dr. Atsushi Miyanohara (UCSD)) and inserted together with approx. 270 bp upstream and downstream sequences of the rat miR-126 pre-miRNA [28] and an IRES-GFP cassette downstream of the miRNA gene into the pRRL.cPPT.WPRE.Sin-18 backbone. The following primers were used to amplify the miRNA sequences from genomic DNA by PCR (given without flanking sequences for restriction sites): rno-miR-126 5′: GCACTATGCTGAGGGCTGATTC; rno-miR-126 3′: TTCTACACCTCCTCTCTCACC. The human sAPPα cDNA was cloned according to the strategy by Turner et al. [47] and inserted into the pRRL.PGK.cPPT.WPRE.Sin-18 backbone. The lentivirus construct CAG.NGF.GFP that expresses NGF from the chicken beta actin promoter with a CMV enhancer element was kindly provided by Dr. I. Verma, Salk Inst. La Jolla, CA [48, 49]. For downregulating trkB expression, a set of four trkB-siRNA-expressing lentivirus vectors was used (TRCN0000023416, TRCN0000023699, TRCN0000023701, TRCN0000023703; The RNAi Consortium (http://www.broadinstitute.org/rnai/public/); Thermo Scientific/Dharmacon (http://dharmacon.gelifesciences.com/openbiosystems)).
All cloning experiments were based on standard molecular biology techniques. Virus production, concentration by ultracentrifugation, and qRT-PCR- or p24 ELISA- (Clontech Laboratories, Mountain View, CA) based titer determination were performed according to published protocols [50–52]. Average virus titers were 106–107 transducing units per μl.
Cell transductions were performed with multiplicity of infections (MOI) of 10–20 in the presence of 5–7 μg/ml hexadimethrine bromide (Polybrene, Sigma Aldrich, St. Louis, MO). Cells were incubated with virus and polybrene for 5–6 h before changing to fresh media. Expression of virus vectors was determined by GFP fluorescence and qRT-PCR using the miR-126 TaqMan® MicroRNA Assay from Life Technologies Corporation (Cat. no. 4427975) and rat snoRNA (Cat. no. U64702) or the Exiqon mmu-mir-126-5p and RNU5G control (Exiqon, Woburn, MA).
Animals and Primary Cell Culture
All procedures involving animals were approved by the IACUC committees at McLean Hospital or Hanyang University. Primary cortical or hippocampal neurons were obtained from embryonic day 18 (E18) rat embryos (Sprague-Dawley, Charles River, MA), Tg6799 transgenic (MT) mice (Jackson Laboratory, Bar Harbor, ME), or littermate controls (LM) at postnatal day 1 as described [53, 28]. Briefly, dissected cortex and hippocampus brain tissues were incubated in Accutase (Invitrogen) for 10 min at 37 °C and then mechanically triturated using fire-polished Pasteur pipettes. The cell suspension was plated onto glass coverslips in 24-well plates precoated with 37.5 μg/ml poly-d-lysin or poly-l-ornithine (Sigma), 2.5 μg/ml fibronectin (Sigma) or laminin (Sigma) at a density of 3.6 × 104 cells/cm2 in neurobasal media (neurobasal media, 1 % heat-inactivated FBS, penicillin, streptomycin, B27 supplement, glutamax, 2 mg/ml glutamic acid, and additional 1 % horse serum for cortical cells (Invitrogen)) or DMEM/F-12 supplemented with 10 % fetal bovine serum (GenDEPOT, San Diego, CA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis, MO), 2 mM l-glutamine (Gibco, Carlsbad, CA), 5 % B-27 supplement (Gibco, Carlsbad, CA), and 10 ng/ml bFGF (Invitrogen, Carlsbad, CA). Neurobasal media was half-changed with neural differentiation media (neurobasal media without serum and glutamic acid) 4 days after plating to induce cholinergic differentiation, and changed once a week. Cells were transduced with lentiviruses 6 days after plating, and media was half-changed with insulin-free media 5 days after transduction. Dox-inducible miR-126 expressing PC12 cell lines were used for miR-126 inhibition assays by transfection with 50–100 nM scrambled controls or miR-126 targeting locked nucleic acids (LNATM, Exiqon, Woburn, MA) using lipofectamine (Life Technologies, Rockville, MD) as previously described [28].
Drug Treatment and Measurement of Cell Viability
Neuronal cultures were maintained in insulin-free media for 6 days before treatment with STS (Cayman Chemical Company, Ann Arbor, MI), Aβ1–42 (AnaSpec Inc., Fremont, CA), IGF-1 (Peprotech, Rocky Hill, NJ), BDNF (Peprotech), or the IGF-1R tyrosine kinase inhibitor AG1024 (Millipore, Tenecula, CA). STS was dissolved in DMSO (100 μM stock solution) and applied for 24 h at a final concentration of 0, 25, 50, 100, or 300 nM in the primary cultures or PC12 cells, respectively. Lyophilized Aβ1–42 peptides were dissolved in 1 % NH4OHM and then immediately diluted with 1× Dulbecco’s phosphate-buffered saline (PBS) without MgCl2 and CaCl2 (Gibco/Life Technologies #14200-075), as a 150 μM final stock solution for stabilization. The Aβ1–42 stock solution was incubated at 37 °C for 72 h to produce oligomers. For titration, cells were treated with 0, 0.1, 1, 2, or 10 μM Aβ1–42 oligomers for 48–72 h; 1 μM of the final concentration was applied in primary cultures and 2 μM in PC12 cells. Formation of toxic Aβ1–42 oligomers were confirmed in Western blots and toxicity titration assays. IGF-1 (20 ng/ml) was added 30 min before the addition of STS or Aβ1–42. AG1024 was prepared as 2 mM stock solution in DMSO and 0.5 μM of the final concentration was added 30 min prior to IGF-1 treatment.
Cell viability was determined using the activity of lactate dehydrogenase (LDH) in collected cell culture medium, according to the manufacturer’s instructions (Roche, Indianapolis, IN), and absorbance measured at 490 nm.
Protein Sample Preparation and Western Blot
Protein samples were purified from harvested cells in lysis buffer (100 mM Tris-HCl (pH 7. 5), 10 mM EDTA, 10 mM EGTA, 1 % SDS, 20 mM NaCl (Sigma, St. Louis, MO)) containing 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA). Lysates were centrifuged at 14,000 rpm for 30 min at 4 °C and the supernatants collected and stored at −80 °C before use. Equal amounts of protein sample were used for Western blots as previously described [50, 28]. Western blots were performed with the following primary antibodies: PI3-kinase p85β (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1250); IRS-1, AKT, phospho-AKT, ERK, phospho-ERK, GSK-3β, and phospho-GSK-3β (Cell Signaling, 1:1250); 6E10 (Covance, Princeton, NJ, 1:1000); 22C11 (Millipore, 1:1000); and β-actin (Covance, 1:10,000). Alkaline phosphatase (AP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen, 1:2500) and Immun-StarTM AP substrate (Bio-Rad, Hercules, CA) were used for protein detection. sAPPα in the media from PGK.sAPPα-transduced PC12 cells was measured in Western blots using the 22C11 or 6E10 antibody. Quantification of immunoreactive bands was performed using Image J (NIH, http://rsb.info.nih.gov/ij/). Experiments were performed at least in triplicate for the same samples.
Immunocytochemistry
Cultured neurons were fixed in 4 % paraformaldehyde (Fisher Scientific, Waltham, MA) and rinsed with PBS. Cells were then incubated with blocking buffer (10 % normal goat serum and 0.1 % Triton X-100) for 30 min at room temperature. Immunostaining was performed using primary antibodies against choline acetyltransferase (ChAT; Millipore, Tenecula, CA, 1:500), β-III tubulin (Tuj1; Covance, 1:1000), or Tau (Tau46, Cell Signaling, 1:500), followed by incubation in Alexa Fluor 568- or Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen, 1:1000), or alkaline phosphate substrate solution (Vector Lab, Burlingham, CA). After counterstaining with 1 μg/ml Hoechst 33342 (Sigma) for 2 min, cover glasses were mounted onto glass slides using Gel-Mount anti-fade media (Electron Microscopy Sciences, Hatfield, PA).
Cell Counting and Neurite Length Measurement
Neurons were counted from images taken with an inverted Zeiss Axiovision microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) connected to a fluorescence light source and digital camera (Zeiss AxioCam HRc). In each condition, 30 sections per coverslip were quantified and a total of 300–1400 cells were analyzed. Two investigators, blinded to the treatment groups, independently performed counting and duplicate analyses. Neurite lengths of Tau-positive cells were counted from 16 microscopic images per condition and analyzed using Image J (NIH, http://rsb.info.nih.gov/ij/) by two independent assessors blinded to the conditions. The length of neurites was measured using the freehand line tool by drawing lines starting from the basal line of the cell surface to the end of the neurite projection on the image. Each protruding tip from the basal line of the soma was counted as an individual neurite (83–114 cells and 300 neurites per group). The lengths and counts of neurites were presented as a relative value compared to untreated LM control group.
Statistical Analysis
Microsoft Excel software (Microsoft Corp., Redmond, WA) was used for statistical analyses. Data were compared between different experimental groups or within a group using unpaired two-tailed Student’s t test. Differences of comparison were considered statistically significant when p values were less than 0.05 (p < 0.05).
Results
Overexpression of MiR-126 Increases STS Toxicity and Decreases the Neuroprotective Effects of IGF-1
To express GFP or miR-126 together with GFP specifically in neurons, we used lentivirus vectors that contain the Synapsin promoter (Syn.GFP and Syn.miR-126, respectively) (Fig. 1a, b). Virus-transduced cortical or hippocampal primary cultures were tested in STS or Aβ1–42 toxicity assays (Supplementary Material Fig. S1a–c) in combination with trophic factors expressed from viral vectors (CAG.NGF [48, 49] and PGK.sAPPα (Supplementary Material Fig. S1d)) or were supplemented to the cultures (IGF-1 and BDNF).
We first tested the effects of miR-126 on toxicity to STS, which causes a general inhibition of protein kinase activities and whose effects can be ameliorated by IGF-1 [40, 41]. Overexpression of miR-126 increased STS toxicity and reduced the protective effects of IGF-1 when compared to naïve and virus GFP controls (Fig. 1c). The effect of IGF-1 was inhibited in the presence of the IGF-1 receptor (IGF-1R) inhibitor AG1024 in both control and miR-126-transduced cells, confirming that miR-126 acts on IGF-1 signaling pathways [54, 20, 55, 28] (Fig. 1d). To confirm apoptotic neuronal cell death measured by LDH, we immunostained the cultures with β-III tubulin and the nuclear marker Hoechst 33342. Apoptotic neurons were characterized by swollen cytoplasm and condensed or fragmented nuclei (Supplementary Material Fig. S2). The percent of apoptotic neurons over all apoptotic cells was increased in Syn.miR-126-transduced cultures, demonstrating neuron specificity of the miR-126 effects. When we examined miR-126 expression levels, we found that the endogenous miRNA was increased (1.5–2-fold) in STS- and IGF-1-treated cells (Fig. 1e), while in the Syn.miR-126-transduced cells, STS caused a decrease and IGF-1 a slight increase in miRNA levels, which could have been associated with different Synapsin promoter regulation as a consequence of cell treatment.
Overexpression of MiR-126 Is Neurotoxic, Increases Aβ1–42 Toxicity, and Modulates Neuroprotection by IGF-1, NGF, BDNF, and sAPPα
We next focused on other factors that are associated with PI3K or ERK signaling and which are involved in the neuronal aging process or disease-specific pathogenesis. Aβ1–42, which is thought to be the primary toxic agent in AD pathogenesis [37, 35], acts on the insulin/IGF-1 or NGF receptor and, thus, competes with insulin/IGF-1 or NGF on PI3K signal activation [34]. In contrast, sAPPα can act synergistically with NGF and IGF-1 to reverse amyloid Aβ toxicity [36, 34]. We, therefore, tested the effects of overexpressed miR-126 on toxic Aβ1–42 peptides and neuroprotection by IGF-1, NGF, and sAPPα in Aβ1–42 vulnerable cell types, including cortical and hippocampal neurons (Figs. 2 and S3). No differences between naïve and control Syn.GFP-transduced cells were observed regarding Aβ1–42 toxicity and the effects of trophic factors (Supplementary Material Fig. S3a and b). In the miR-126 overexpressing neurons, the miRNA alone was cell toxic and exaggerated Aβ1–42 toxicity (Fig. 2a, Supplementary Material Fig. S3c). IGF-1, NGF, and sAPPα reduced Aβ1–42 toxicity in both virus controls and miR-126 overexpressing cells and sAPPα acted synergistically with IGF-1 and NGF, and the effects of IGF-1 in Syn.GFP control, Syn.miR-126, and Syn.miR-126/PGK.sAPPα transduced cells could be inhibited in presence of AG1024 (Fig. 2b). Assessment of miRNA expression levels revealed no marked changes in the virus-transduced cells and factor-treated cells (Fig. 2c, d).
We also tested the effects of miR-126 overexpression in BDNF-treated cortical neurons, because recent data have shown that BDNF protects cortical neurons from Aβ toxicity [56]. BDNF had a neuroprotective effect toward Aβ1–42 toxicity in both Syn.GFP controls and Syn.miR-126-transduced cells, but could not fully abrogate the increased neurotoxicity caused by miR-126 (Fig. 3a). In both conditions, the neuroprotective effects of BDNF were diminished when the expression of its receptor trkB was inhibited by trkB-siRNAs, demonstrating that miR-126 affects the BDNF/trkB signaling cascade (Fig. 3a, b).
MiR-126 Increases Aβ1–42 Toxicity in Tg6799 Neurons and Modulates Neurite Sprouting
We next evaluated the effects of miR-126 in primary cortical cultures from Tg6799 mutant mice because these animals exhibit a massive accumulation of Aβ1–42 in the brain [44]. Overexpression of miR-126 increased Aβ1–42 toxicity in both littermate controls (LM) and Tg6799 mutant (MT) cells and to a greater extent in the latter cell population (Fig. 4a). As shown in the rat primary cultures (Fig. 2), endogenous miR-126 expression was not significantly upregulated in LM neurons after Aβ1–42 treatment, but increased in Tg6799 cells (Fig. 4b).
In addition to neurotoxicity, we also evaluated the effects of miR-126 on neurite sprouting in Tau-immunostained LM control and Tg6799 MT neurons. In the LM cells, Aβ1–42 slightly decreased the length of neurites per cell and this effect was significantly exaggerated when miR-126 was overexpressed (Fig. 4c). Untreated Tg6799 neurons had a reduction in neurite lengths when compared to LM controls, and both Aβ1–42 treatment and miR-126 overexpression significantly increased neurite lengths to levels seen in the untreated LM controls, however, to a lesser extent in Aβ1–42-treated miR-126 overexpressing cells (Fig. 4c). These data indicate that in normal neurons, elevated levels of miR-126 exaggerate an inhibitory effect of Aβ1–42 on neurite sprouting, while in Tg6799 neurons, miR-126 promotes neurite sprouting to a similar extent as seen with Aβ1–42 but has no synergistic effect.
Increased Levels of MiR-126 Affect the Expression of Factors in IGF-1/PI3K/GSK-3β and ERK Signaling
To evaluate the effects of increased miR-126 on cellular signaling events in STS or Aβ1–42 toxicity, we measured the expression of factors in the IGF-1/PI3K/AKT and ERK pathways (Figs. 5 and 6, Supplementary Material Fig. S4), including GSK-3β, which is regulated by pAKT and has been linked to Tau phosphorylation, amyloid production, and neuronal death [57].
In STS toxicity, IRS-1 the adapter molecule of IGF-1R and a validated target of miR-126 [20, 58] was upregulated in controls when exposed to IGF-1, but its expression was reduced in the miR-126 overexpressing cells (Fig. 5a, b). In IGF-1-untreated controls, STS treatment did not change the expression levels of AKT, ERK, and GSK-3β, but caused a downregulation of pAKT, pERK, and pGSK-3β and altered their respective ratios (Fig. 5a, c). The addition of IGF-1 increased AKT, pAKT, pERK, and pGSK-3β, but not ERK and GSK-3β. In contrast, overexpression of miR-126 in STS- and IGF-1-treated neurons caused downregulation of AKT, ERK, GSK-3β, pAKT, pERK, and pGSK-3β, an upregulation of the AKT/pAKT and ERK/pERK ratios, while the pGSK-3β/GSK-3β ratio in the IGF-1 condition was decreased.
In Aβ1–42 toxicity, IGF-1, NGF, and sAPPα caused upregulation of IRS-1 in control neurons but downregulation when miR-126 was overexpressed (Fig. 6a, b, Supplementary Material Fig. S4a). The expression levels of p85β, a component of the PI3K complex and another validated target of miR-126 [55, 59, 54], were also increased in the controls, but unchanged or only slightly increased in the miR-126-transduced cells (Fig. 6a, b, Supplementary Material Fig. S4a). In controls, Aβ1–42 and GF treatment were associated with the upregulation of AKT, pAKT, ERK, pERK, and GSK-3β and to a lesser extent pGSK-3β (Fig. 6a, c, Supplementary Material Fig. S4b). In contrast, except for an upregulation of pERK and pGSK-3β, these molecules were downregulated in miR-126 overexpressing neurons. Moreover, in Aβ1–42 and factor-treated controls, the pAKT/AKT ratios were largely unchanged, while the pERK/ERK and pGSK-3β/GSK-3β ratios were reduced. In contrast, miR-126 overexpression caused a slight decrease of the pAKT/AKT ratios and a striking increase in the pERK/ERK and pGSK-3β/GSK-3β ratios. Altogether, overexpression of miR-126 in neurons had profound impacts on the activation status of signaling pathways related to IGF-1, NGF, and sAPPα in STS and Aβ1–42 toxicity.
Inhibition of MiR-126 Is Neuroprotective
Finally, we evaluated whether inhibition of miR-126 would be neuroprotective to STS and Aβ1–42 toxicity. For this, we used a toxicity assay based on virus-transduced PC12 cells as previously published in [28]. Consistent with our data on 6-OHDA, inhibition of miR-126 reduced STS toxicity and enhanced the neuroprotective effects of IGF-1 (Fig. 7a) and also diminished the toxic effects of Aβ1–42 (Fig. 7b).
Discussion
Neuronal functions depend on a balance between neurotoxicity and neuroprotection, with the latter mediated in part by GF/PI3K signaling pathways. In aging and age-related neurological diseases, slow progressive neuronal dysfunction is a consequence of an imbalance of many mechanisms, which may be general or disease specific. Because of its implication in aging and neurodegenerative diseases, insulin/IGF-1 signaling is one of the pathways of great interest. For example, there is evidence that AD may be a metabolic disorder with an impairment of glucose utilization and energy production, as a consequence of insulin deficiency and resistance to insulin/IGF-1/PI3K signaling in the brain (sometimes called “brain-diabetes” or “type-3-diabetes”) [2–8, 60–62]. Dysfunctional insulin/IGF-1 signaling contributes to all aspects of AD-type neurodegeneration, including dysregulated Αβ, Tau hyperphosphylation, and oxidative stress [8, 63–67, 35]. The PI3K signaling pathway is also used by other GFs, including BDNF and NGF, and there is evidence that resistance to NGF signaling contributes to the loss of cholinergic neurons and cognitive decline seen both in aging and dementia [30–33]. The molecular mechanisms underlying these disturbances are largely unknown, and our data provide evidence that miR-126 may play a role in these processes.
MiR-126 is involved in regulating IGF-1/PI3K/AKT, p38 MAPK, or ERK signaling in a multitude of nonneuronal cells [14, 54, 19, 68–70]. In the neuronal context, it is expressed in rodent or human cortical, hippocampal, cerebellum, ventral mesencephalon, and motor neurons [21, 22, 26, 23, 27], and the miRNA is differentially expressed in the cortex, hippocampus, and cerebellum during early human development [27]. Recently, we found an upregulation of miR-126 in postmortem DA neurons from PD and in pyramidal cortical neurons from schizophrenia patients [14, 28, 29], and in DA cell systems, elevated levels of miR-126 increased neurotoxic to 6-OHDA by downregulating IGF-1/PI3K and ERK signaling [28]. Our new finding that miR-126 also increases STS and Aβ1–42 toxicity in cortical and hippocampal neurons and diminishes the protective effects of a variety of growth factors suggests that miR-126 could be involved in the general survival mechanisms of neurons and that its deregulation could contribute to neuronal dysfunction in aging and in combination with cell- and disease-specific events to neurodegeneration. Thus, miR-126 may be an important mechanistic link between metabolic dysfunction and neurotoxicity.
In AD pathogenesis, there is evidence that insulin/IGF-1 affects Aβ metabolism and function, e.g., stimulating its trafficking from the Golgi apparatus, its extracellular secretion, and increasing transcription of Aβ degrading proteins [63–67, 35]. In turn, Aβ acts on the insulin/IGF-1 or NGF receptor and, thus, competes with insulin/IGF-1 or NGF on PI3K signal activation [34] (Fig. 8). Aβ appears to alter insulin/IGF-1 signaling by inappropriately increasing the activation of PI3K/AKT/mammalian target of rapamycin (mTOR) and JNK signaling and feedback inhibition of normal activation, thereby reducing normal insulin/IGF-1 functions, including normal on/off switching and the protective effects of FOXO activation and mTOR inactivation [43]. Also, intracellular Aβ appears to directly interfere with PIK3 activation of AKT and subsequent GSK-3β phosphorylation which is partly responsible for Tau hyperphosphorylation and the regulation of Tau gene expression [71–73, 57, 74, 34, 42]. In contrast, as shown in our study and previously demonstrated by Luo et al. [36] and Jimenez et al. [34], sAPPα acts synergistically with NGF or IGF-1 and reverses amyloid Aβ toxicity. Similarly, a neuroprotective role of BDNF against Aβ toxicity in cortical neurons [56] was also observed in our study. The finding that elevated levels of miR-126 exaggerate Aβ toxicity in both GF-untreated and protective conditions suggests that dysfunctional miR-126 may be a central factor in its pathogenesis via specifically deregulating PI3K/AKT signaling cascades (Fig. 8). This notion is corroborated by the observation that cortical neurons from Tg6799 mice had elevated miR-126 and an increase in Aβ toxicity. Both, miR-126 or Aβ alone, or in combination, negatively affected neurite sprouting in littermate control neurons, but increased sprouting in the cells with FAD-associated mutations. FAD mutations seem to have inhibiting or promoting effects on neurite growth and plasticity, and the latter has been associated with increased GSK-3β activity and Aβ1–24-induced phosphorylation of Tau [75, 76]. However, the exact role that miR-126 plays in these functions needs further investigation.
A series of miR-126 targets has been described (summarized in [14]), including factors in ERK signaling, which was also downregulated in the miR-126 overexpressing neurons. One of these targets is SPRED1 [54, 28], which inhibits MAPK/ERK signaling, and this pathway is involved in neuronal cell function, aging, and degeneration, including Tau regulation [77]. Recently, delta-like 1 homologue (Dlk1), an epidermal growth factor-like homeotic protein that can control extracellular IGF-1 levels by binding IGF-binding protein 1 (IGFBP1)/IGF-1 complexes [78], was identified as a novel target of miR-126 [79]. Dlk1 activates the MEK/ERK pathway [80], plays a functional role in motor neurons [81], and has been identified as a novel target of the orphan nuclear receptor Nurr1 in meso-diencephalon DA neurons [82]. MiR-126 may also not exclusively regulate PI3K signaling. For example, in the hippocampus of aged Ames dwarf and growth hormone receptor knockout mice, miR-470, miR-669b, and miR-681 were identified as potential suppressors of IGF-1R and AKT [17]; in glioblastoma cells, miR-7 inhibits IGF-1/AKT signaling by targeting IRS-1 [83]; and in transfected N2A cells that stably express APP, overexpression of miR-98 downregulates its target IGF-1 and indirectly increases Aβ production and Tau phosphorylation [84]. In addition, miR-320, which is expressed in neurons, including pigmented neurons in the substantia nigra [22], influences IGF-1 signaling through regulation of IGF-1/2, IGF-1R, phosphoinositide-3-kinase regulatory subunit 1 (p85α; PIK3R1), and the glucose transporter 4 (SLC2A4) [85, 86].
Although levels of miR-126 appear to be low in neurons, small increases seem to have striking effects on cell function. Our observation that neurotoxicity is a consequence of 4-fold upregulated miR-126 is consistent with findings in hepatocytes, in which rotenone-induced dysfunctional mitochondria caused insulin resistance that was associated with a 3- to 4-fold increase of miR-126, a 75 % decrease of IRS-1, an insulin-induced reduction of glycogen, and downregulation of the pAKT/AKT and pGSK-3β/GSK3-3β ratios [20]. Together, these data indicate that small changes of miR-126 levels could have profound effects on cell function pointing to a potential potent role of this miRNA in fine-tuning and balancing (or dysbalancing) GF/PI3K signaling in neurons. In fact, inhibition of miR-126 is neuroprotective and increases the neuroprotective effects of GFs without seemingly compromising normal neuronal cell function. Given its low expression levels, this could indicate that miR-126 may be dispensable for normal cellular homeostasis, while being detrimental when upregulated in the context of neuronal insult. This notion is supported from studies on miR-126 k.o. mice [87, 79]. While about 40 % of mice die embryonically or perinatally with severe vascular abnormalities, surviving animals appear to be normal with no reported vascular defects or brain damage, supporting the hypothesis that miR-126 may be dispensable in adult brain cells. Therefore, diminishing or eliminating miR-126’s function may be a strategy to promote GF activities. The single locus of miR-126 in intron 7 of the EGFL7 gene makes it an optimal target for gene editing, since its targeted deletion does not alter the expression of EGFL7 in homozygous transgenic mice [87].
In summary, our results provide evidence for a novel mechanism of regulating GF/PI3K and ERK signaling by miR-126 in neurons and suggest that GF/pathway deregulation by dysfunctional miR-126 may be a contributing mechanism in the resistance of neurons to GF signaling events during aging and in the pathogenesis of neurodegenerative diseases, such as PD and AD. While small increases in miR-126 increase neuronal vulnerability to toxic insult in both normal cells and seemingly augmented in cells with disease-associated mutations, such as in FAD, inhibiting miR-126 confers neuroprotection without compromising normal neuronal cell functions, suggesting that nonfunctional or dispensable miR-126 in neurons may have therapeutic potential.
References
Bassil F, Fernagut PO, Bezard E, Meissner WG (2014) Insulin, IGF-1 and GLP-1 signaling in neurodegenerative disorders: targets for disease modification? Prog Neurobiol 118C:1–18. doi:10.1016/j.pneurobio.2014.02.005
Craft S, Watson GS (2004) Insulin and neurodegenerative disease: shared and specific mechanisms. Lancet Neurol 3(3):169–178
Deak F, Sonntag WE (2012) Aging, synaptic dysfunction, and insulin-like growth factor (IGF)-1. J Gerontol A Biol Sci Med Sci 67(6):611–625. doi:10.1093/gerona/gls118
Talbot K, Wang HY, Kazi H, Han LY, Bakshi KP, Stucky A, Fuino RL, Kawaguchi KR, Samoyedny AJ, Wilson RS, Arvanitakis Z, Schneider JA, Wolf BA, Bennett DA, Trojanowski JQ, Arnold SE (2012) Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest 122(4):1316–1338. doi:10.1172/JCI59903
Torres-Aleman I (2010) Toward a comprehensive neurobiology of IGF-I. Dev Neurobiol 70(5):384–396
Kim B, Feldman EL (2012) Insulin resistance in the nervous system. Trends Endocrinol Metab 23(3):133–141. doi:10.1016/j.tem.2011.12.004
Bomfim TR, Forny-Germano L, Sathler LB, Brito-Moreira J, Houzel JC, Decker H, Silverman MA, Kazi H, Melo HM, McClean PL, Holscher C, Arnold SE, Talbot K, Klein WL, Munoz DP, Ferreira ST, De Felice FG (2012) An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease-associated Abeta oligomers. J Clin Invest 122(4):1339–1353. doi:10.1172/JCI57256
de la Monte SM, Tong M (2014) Brain metabolic dysfunction at the core of Alzheimer’s disease. Biochem Pharmacol 88(4):548–559. doi:10.1016/j.bcp.2013.12.012
Muller AP, Fernandez AM, Haas C, Zimmer E, Portela LV, Torres-Aleman I (2012) Reduced brain insulin-like growth factor I function during aging. Mol Cell Neurosci 49(1):9–12. doi:10.1016/j.mcn.2011.07.008
Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355
Heman-Ackah SM, Hallegger M, Rao MS, Wood MJ (2013) RISC in PD: the impact of microRNAs in Parkinson’s disease cellular and molecular pathogenesis. Front Mol Neurosci 6:40. doi:10.3389/fnmol.2013.00040
Ma L, Wei L, Wu F, Hu Z, Liu Z, Yuan W (2013) Advances with microRNAs in Parkinson’s disease research. Drug Des Devel Ther 7:1103–1113. doi:10.2147/DDDT.S48500
Sonntag KC, Wahlestedt C (2010) RNA mechanisms in CNS systems and disorders. Brain Res 1338:1–2
Sonntag KC, Woo TU, Krichevsky AM (2012) Converging miRNA functions in diverse brain disorders: a case for miR-124 and miR-126. Exp Neurol 235(2):427–435. doi:10.1016/j.expneurol.2011.11.035
Du L, Pertsemlidis A (2011) Cancer and neurodegenerative disorders: pathogenic convergence through microRNA regulation. J Mol Cell Biol 3(3):176–180
Li N, Bates DJ, An J, Terry DA, Wang E (2011) Up-regulation of key microRNAs, and inverse down-regulation of their predicted oxidative phosphorylation target genes, during aging in mouse brain. Neurobiol Aging 32(5):944–955. doi:10.1016/j.neurobiolaging.2009.04.020
Liang R, Khanna A, Muthusamy S, Li N, Sarojini H, Kopchick JJ, Masternak MM, Bartke A, Wang E (2011) Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice. Aging Cell 10(6):1080–1088. doi:10.1111/j.1474-9726.2011.00751.x
Persengiev SP, Kondova II, Bontrop RE (2012) The impact of microRNAs on brain aging and neurodegeneration. Curr Gerontol Geriatr Res 2012:359369. doi:10.1155/2012/359369
Fish JE, Srivastava D (2009) MicroRNAs: opening a new vein in angiogenesis research. Sci Signal 2 (52):pe1. doi:10.1126/scisignal.252pe1
Ryu HS, Park SY, Ma D, Zhang J, Lee W (2011) The induction of microRNA targeting IRS-1 is involved in the development of insulin resistance under conditions of mitochondrial dysfunction in hepatocytes. PLoS One 6(3):e17343
Wei H, Wang C, Zhang C, Li P, Wang F, Zhang Z (2010) Comparative profiling of microRNA expression between neural stem cells and motor neurons in embryonic spinal cord in rat. Int J Dev Neurosci 28(6):545–551
Nelson PT, Wang WX, Rajeev BW (2008) MicroRNAs (miRNAs) in neurodegenerative diseases. Brain Pathol 18(1):130–138
Nunez-Iglesias J, Liu CC, Morgan TE, Finch CE, Zhou XJ (2010) Joint genome-wide profiling of miRNA and mRNA expression in Alzheimer’s disease cortex reveals altered miRNA regulation. PLoS One 5(2):e8898. doi:10.1371/journal.pone.0008898
Hebert SS, Horre K, Nicolai L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B (2008) Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A 105(17):6415–6420
Wang WX, Huang Q, Hu Y, Stromberg AJ, Nelson PT (2011) Patterns of microRNA expression in normal and early Alzheimer’s disease human temporal cortex: white matter versus gray matter. Acta Neuropathol 121(2):193–205. doi:10.1007/s00401-010-0756-0
Miller BH, Zeier Z, Xi L, Lanz TA, Deng S, Strathmann J, Willoughby D, Kenny PJ, Elsworth JD, Lawrence MS, Roth RH, Edbauer D, Kleiman RJ, Wahlestedt C (2012) MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function. Proc Natl Acad Sci U S A 109(8):3125–3130. doi:10.1073/pnas.1113793109
Ziats MN, Rennert OM (2014) Identification of differentially expressed microRNAs across the developing human brain. Mol Psychiatry 19(7):848–852. doi:10.1038/mp.2013.93
Kim W, Lee Y, McKenna ND, Yi M, Simunovic F, Wang Y, Kong B, Rooney RJ, Seo H, Stephens RM, Sonntag KC (2014) miR-126 contributes to Parkinson’s disease by dysregulating the insulin-like growth factor/phosphoinositide 3-kinase signaling. Neurobiol Aging 35(7):1712–1721. doi:10.1016/j.neurobiolaging.2014.01.021
Pietersen CY, Mauney SA, Kim SS, Lim MP, Rooney RJ, Goldstein JM, Petryshen TL, Seidman LJ, Shenton ME, McCarley RW, Sonntag KC, Woo TU (2014) Molecular profiles of pyramidal neurons in the superior temporal cortex in schizophrenia. J Neurogenet 28(1–2):53–69. doi:10.3109/01677063.2014.882918
Mufson EJ, Counts SE, Perez SE, Ginsberg SD (2008) Cholinergic system during the progression of Alzheimer’s disease: therapeutic implications. Expert Rev Neurother 8(11):1703–1718
Salehi A, Delcroix JD, Belichenko PV, Zhan K, Wu C, Valletta JS, Takimoto-Kimura R, Kleschevnikov AM, Sambamurti K, Chung PP, Xia W, Villar A, Campbell WA, Kulnane LS, Nixon RA, Lamb BT, Epstein CJ, Stokin GB, Goldstein LS, Mobley WC (2006) Increased App expression in a mouse model of Down’s syndrome disrupts NGF transport and causes cholinergic neuron degeneration. Neuron 51(1):29–42
Cooper JD, Salehi A, Delcroix JD, Howe CL, Belichenko PV, Chua-Couzens J, Kilbridge JF, Carlson EJ, Epstein CJ, Mobley WC (2001) Failed retrograde transport of NGF in a mouse model of Down’s syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion. Proc Natl Acad Sci U S A 98(18):10439–10444
Schulte-Herbruggen O, Jockers-Scherubl MC, Hellweg R (2008) Neurotrophins: from pathophysiology to treatment in Alzheimer’s disease. Curr Alzheimer Res 5(1):38–44
Jimenez S, Torres M, Vizuete M, Sanchez-Varo R, Sanchez-Mejias E, Trujillo-Estrada L, Carmona-Cuenca I, Caballero C, Ruano D, Gutierrez A, Vitorica J (2011) Age-dependent accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model. J Biol Chem 286(21):18414–18425. doi:10.1074/jbc.M110.209718
O’Brien RJ, Wong PC (2011) Amyloid precursor protein processing and Alzheimer’s disease. Annu Rev Neurosci 34:185–204. doi:10.1146/annurev-neuro-061010-113613
Luo JJ, Wallace MS, Hawver DB, Kusiak JW, Wallace WC (2001) Characterization of the neurotrophic interaction between nerve growth factor and secreted alpha-amyloid precursor protein. J Neurosci Res 63(5):410–420
Holtzman DM, Morris JC, Goate AM (2011) Alzheimer’s disease: the challenge of the second century. Sci Transl Med 3 (77):77sr71. doi:10.1126/scitranslmed.3002369
Zuccato C, Cattaneo E (2009) Brain-derived neurotrophic factor in neurodegenerative diseases. Nat Rev Neurol 5(6):311–322. doi:10.1038/nrneurol.2009.54
Autry AE, Monteggia LM (2012) Brain-derived neurotrophic factor and neuropsychiatric disorders. Pharmacol Rev 64(2):238–258. doi:10.1124/pr.111.005108
Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT, Faraoni R, Floyd M, Hunt JP, Lockhart DJ, Milanov ZV, Morrison MJ, Pallares G, Patel HK, Pritchard S, Wodicka LM, Zarrinkar PP (2008) A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 26(1):127–132. doi:10.1038/nbt1358
Ryu BR, Ko HW, Jou I, Noh JS, Gwag BJ (1999) Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insulin and IGF-I. J Neurobiol 39(4):536–546
Lee HK, Kumar P, Fu Q, Rosen KM, Querfurth HW (2009) The insulin/Akt signaling pathway is targeted by intracellular beta-amyloid. Mol Biol Cell 20(5):1533–1544. doi:10.1091/mbc.E08-07-0777
O’Neill C (2013) PI3-kinase/Akt/mTOR signaling: Impaired on/off switches in aging, cognitive decline and Alzheimer’s disease. Exp Gerontol 48(7):647–653. doi:10.1016/j.exger.2013.02.025
Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci : Off J Soc Neurosci 26(40):10129–10140. doi:10.1523/JNEUROSCI.1202-06.2006
Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72:9873–9880
Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15:871–875
Turner PR, Bourne K, Garama D, Carne A, Abraham WC, Tate WP (2007) Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents. J Neurosci Methods 164(1):68–74
Blesch A, Conner J, Pfeifer A, Gasmi M, Ramirez A, Britton W, Alfa R, Verma I, Tuszynski MH (2005) Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons. Mol Ther 11(6):916–925
Nagahara AH, Bernot T, Moseanko R, Brignolo L, Blesch A, Conner JM, Ramirez A, Gasmi M, Tuszynski MH (2009) Long-term reversal of cholinergic neuronal decline in aged non-human primates by lentiviral NGF gene delivery. Exp Neurol 215(1):153–159
Seo H, Sonntag KC, Isacson O (2004) Generalized brain and skin proteasome inhibition in Huntington’s disease. Ann Neurol 56(3):319–328
Seo H, Sonntag KC, Kim W, Cattaneo E, Isacson O (2007) Proteasome activator enhances survival of Huntington’s disease neuronal model cells. PLoS One 2:e238
Sastry L, Johnson T, Hobson MJ, Smucker B, Cornetta K (2002) Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther 9(17):1155–1162. doi:10.1038/sj.gt.3301731
Chung CY, Seo H, Sonntag KC, Brooks A, Lin L, Isacson O (2005) Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 14(13):1709–1725
Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, Ivey KN, Bruneau BG, Stainier DY, Srivastava D (2008) miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell 15(2):272–284
Guo C, Sah JF, Beard L, Willson JK, Markowitz SD, Guda K (2008) The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers. Genes Chromosomes Cancer 47(11):939–946
Guo S, Kim WJ, Lok J, Lee SR, Besancon E, Luo BH, Stins MF, Wang X, Dedhar S, Lo EH (2008) Neuroprotection via matrix-trophic coupling between cerebral endothelial cells and neurons. Proc Natl Acad Sci U S A 105(21):7582–7587. doi:10.1073/pnas.0801105105
Kaidanovich-Beilin O, Woodgett JR (2011) GSK-3: functional insights from cell biology and animal models. Front Mol Neurosci 4:40. doi:10.3389/fnmol.2011.00040
Zhang J, Du YY, Lin YF, Chen YT, Yang L, Wang HJ, Ma D (2008) The cell growth suppressor, mir-126, targets IRS-1. Biochem Biophys Res Commun 377(1):136–140
Zhu N, Zhang D, Xie H, Zhou Z, Chen H, Hu T, Bai Y, Shen Y, Yuan W, Jing Q, Qin Y (2011) Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2. Mol Cell Biochem 351(1–2):157–164
Sato N, Takeda S, Uchio-Yamada K, Ueda H, Fujisawa T, Rakugi H, Morishita R (2011) Role of insulin signaling in the interaction between Alzheimer disease and diabetes mellitus: a missing link to therapeutic potential. Curr Aging Sci 4(2):118–127
Candeias E, Duarte AI, Carvalho C, Correia SC, Cardoso S, Santos RX, Placido AI, Perry G, Moreira PI (2012) The impairment of insulin signaling in Alzheimer’s disease. IUBMB Life 64(12):951–957. doi:10.1002/iub.1098
Moloney AM, Griffin RJ, Timmons S, O’Connor R, Ravid R, O’Neill C (2010) Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer’s disease indicate possible resistance to IGF-1 and insulin signalling. Neurobiol Aging 31(2):224–243. doi:10.1016/j.neurobiolaging.2008.04.002
Watson GS, Peskind ER, Asthana S, Purganan K, Wait C, Chapman D, Schwartz MW, Plymate S, Craft S (2003) Insulin increases CSF Abeta42 levels in normal older adults. Neurology 60(12):1899–1903
Gasparini L, Gouras GK, Wang R, Gross RS, Beal MF, Greengard P, Xu H (2001) Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling. J Neurosci : Off J Soc Neurosci 21(8):2561–2570
Messier C, Teutenberg K (2005) The role of insulin, insulin growth factor, and insulin-degrading enzyme in brain aging and Alzheimer’s disease. Neural Plast 12(4):311–328. doi:10.1155/NP.2005.311
Ling X, Martins RN, Racchi M, Craft S, Helmerhorst E (2002) Amyloid beta antagonizes insulin promoted secretion of the amyloid beta protein precursor. J Alzheimers Dis 4(5):369–374
Pandini G, Pace V, Copani A, Squatrito S, Milardi D, Vigneri R (2013) Insulin has multiple antiamyloidogenic effects on human neuronal cells. Endocrinology 154(1):375–387. doi:10.1210/en.2012-1661
Lechman ER, Gentner B, van Galen P, Giustacchini A, Saini M, Boccalatte FE, Hiramatsu H, Restuccia U, Bachi A, Voisin V, Bader GD, Dick JE, Naldini L (2012) Attenuation of miR-126 activity expands HSC in vivo without exhaustion. Cell Stem Cell 11(6):799–811. doi:10.1016/j.stem.2012.09.001
Huang F, Fang ZF, Hu XQ, Tang L, Zhou SH, Huang JP (2013) Overexpression of miR-126 promotes the differentiation of mesenchymal stem cells toward endothelial cells via activation of PI3K/Akt and MAPK/ERK pathways and release of paracrine factors. Biol Chem 394(9):1223–1233. doi:10.1515/hsz-2013-0107
Bai Y, Bai X, Wang Z, Zhang X, Ruan C, Miao J (2011) MicroRNA-126 inhibits ischemia-induced retinal neovascularization via regulating angiogenic growth factors. Exp Mol Pathol 91(1):471–477. doi:10.1016/j.yexmp.2011.04.016
Rankin CA, Sun Q, Gamblin TC (2008) Pre-assembled tau filaments phosphorylated by GSK-3b form large tangle-like structures. Neurobiol Dis 31(3):368–377. doi:10.1016/j.nbd.2008.05.011
Muyllaert D, Terwel D, Borghgraef P, Devijver H, Dewachter I, Van Leuven F (2006) Transgenic mouse models for Alzheimer’s disease: the role of GSK-3B in combined amyloid and tau-pathology. Rev Neurol (Paris) 162(10):903–907
Schubert M, Brazil DP, Burks DJ, Kushner JA, Ye J, Flint CL, Farhang-Fallah J, Dikkes P, Warot XM, Rio C, Corfas G, White MF (2003) Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation. J Neurosci : Off J Soc Neurosci 23(18):7084–7092
Xie L, Helmerhorst E, Taddei K, Plewright B, Van Bronswijk W, Martins R (2002) Alzheimer’s beta-amyloid peptides compete for insulin binding to the insulin receptor. The Journal of neuroscience : the official journal of the Society for Neuroscience 22 (10):RC221. doi:20026383
Alpar A, Ueberham U, Bruckner MK, Seeger G, Arendt T, Gartner U (2006) Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice. Brain Res 1099(1):189–198. doi:10.1016/j.brainres.2006.04.109
Pigino G, Pelsman A, Mori H, Busciglio J (2001) Presenilin-1 mutations reduce cytoskeletal association, deregulate neurite growth, and potentiate neuronal dystrophy and tau phosphorylation. J Neurosci : Off J Soc Neurosci 21(3):834–842
Lee G, Leugers CJ (2012) Tau and tauopathies. Prog Mol Biol Transl Sci 107:263–293. doi:10.1016/B978-0-12-385883-2.00004-7
Nueda ML, Garcia-Ramirez JJ, Laborda J, Baladron V (2008) dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells. J Mol Biol 379(3):428–442. doi:10.1016/j.jmb.2008.03.070
Schober A, Nazari-Jahantigh M, Wei Y, Bidzhekov K, Gremse F, Grommes J, Megens RT, Heyll K, Noels H, Hristov M, Wang S, Kiessling F, Olson EN, Weber C (2014) MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med 20(4):368–376. doi:10.1038/nm.3487
Sul HS (2009) Minireview: Pref-1: role in adipogenesis and mesenchymal cell fate. Mol Endocrinol 23(11):1717–1725. doi:10.1210/me.2009-0160
Muller D, Cherukuri P, Henningfeld K, Poh CH, Wittler L, Grote P, Schluter O, Schmidt J, Laborda J, Bauer SR, Brownstone RM, Marquardt T (2014) Dlk1 promotes a fast motor neuron biophysical signature required for peak force execution. Science 343(6176):1264–1266. doi:10.1126/science.1246448
Jacobs FM, van der Linden AJ, Wang Y, von Oerthel L, Sul HS, Burbach JP, Smidt MP (2009) Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons. Development 136(14):2363–2373. doi:10.1242/dev.037556
Kefas B, Godlewski J, Comeau L, Li Y, Abounader R, Hawkinson M, Lee J, Fine H, Chiocca EA, Lawler S, Purow B (2008) microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res 68(10):3566–3572. doi:10.1158/0008-5472.CAN-07-6639
Hu YK, Wang X, Li L, Du YH, Ye HT, Li CY (2013) MicroRNA-98 induces an Alzheimer’s disease-like disturbance by targeting insulin-like growth factor 1. Neurosci Bull 29(6):745–751. doi:10.1007/s12264-013-1348-5
Wang XH, Qian RZ, Zhang W, Chen SF, Jin HM, Hu RM (2009) MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats. Clin Exp Pharmacol Physiol 36(2):181–188
Ling HY, Ou HS, Feng SD, Zhang XY, Tuo QH, Chen LX, Zhu BY, Gao ZP, Tang CK, Yin WD, Zhang L, Liao DF (2009) CHANGES IN microRNA (miR) profile and effects of miR-320 in insulin-resistant 3T3-L1 adipocytes. Clin Exp Pharmacol Physiol 36(9):e32–e39. doi:10.1111/j.1440-1681.2009.05207.x
Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, Richardson JA, Bassel-Duby R, Olson EN (2008) The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 15(2):261–271
Acknowledgments
We want to thank Dr. D. Trono (EPFL) for providing the third generation lentivirus vector system, Dr. A. Miyanohara (UCSD) for the Synapsin.GFP, and Dr. I. Verma for the CAG.NGF.GFP lentivirus construct. This research was supported by the National Institute of Health grants NS067335 (K.C.S.) and NS070577 (K.S.K.) and by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (NRF Grants 2011–0030928, 2011–0030049, 2012–003338 to H.S.).
Conflict of Interest
The authors have no conflicts of interest.
Authors’ Contributions
K.C.S. was involved in the conception, design, data acquisition, analysis, interpretation, presentation, and write-up for publication; W.K., H.N., Y.L., J.J, A.S., D.L.M., and H.S. were involved with experimental design, data acquisition, analysis, interpretation, presentation, and manuscript preparation; K.S.K. and B.M.C. were involved in manuscript preparation and for additional resources.
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Kim, W., Noh, H., Lee, Y. et al. MiR-126 Regulates Growth Factor Activities and Vulnerability to Toxic Insult in Neurons. Mol Neurobiol 53, 95–108 (2016). https://doi.org/10.1007/s12035-014-8989-x
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DOI: https://doi.org/10.1007/s12035-014-8989-x