MicroRNA-132 provides neuroprotection for tauopathies via multiple signaling pathways
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MicroRNAs (miRNA) regulate fundamental biological processes, including neuronal plasticity, stress response, and survival. Here, we describe a neuroprotective function of miR-132, the miRNA most significantly downregulated in neurons in Alzheimer’s disease. We demonstrate that miR-132 protects primary mouse and human wild-type neurons and more vulnerable Tau-mutant neurons against amyloid β-peptide (Aβ) and glutamate excitotoxicity. It lowers the levels of total, phosphorylated, acetylated, and cleaved forms of Tau implicated in tauopathies, promotes neurite elongation and branching, and reduces neuronal death. Similarly, miR-132 attenuates PHF-Tau pathology and neurodegeneration, and enhances long-term potentiation in the P301S Tau transgenic mice. The neuroprotective effects are mediated by direct regulation of the Tau modifiers acetyltransferase EP300, kinase GSK3β, RNA-binding protein Rbfox1, and proteases Calpain 2 and Caspases 3/7. These data suggest miR-132 as a master regulator of neuronal health and indicate that miR-132 supplementation could be of therapeutic benefit for the treatment of Tau-associated neurodegenerative disorders.
KeywordsAlzheimer’s disease Tauopathies Neurodegeneration Neuroprotection MicroRNA Non-coding RNA
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder typified by profound synaptic loss, brain atrophy, and the presence of extracellular plaques composed of amyloid β-protein (Aβ), and intracellular neurofibrillary tangles (NFTs) formed by hyperphosphorylated Tau [3, 27]. NFTs are also pathogenomic for a range of disorders in which Tau deposits occur in the absence of plaques. The major primary tauopathies include: frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Pick’s disease, and corticobasal degeneration. New therapeutic strategies and targets are desperately needed to treat these devastating diseases.
miRNAs are small regulatory molecules that post-transcriptionally repress gene expression and thereby regulate diverse biological processes, including neuronal differentiation, plasticity, survival, and regeneration . miRNAs are often considered as determinants of cell fate and are also increasingly acknowledged as prime regulators involved in various brain pathologies ranging from neurodevelopmental disorders to brain tumors, to neurodegenerative diseases . Given its immense complexity, the brain expresses the richest repertoire of miRNA species, with specific miRNAs being highly enriched in certain cell types of the brain, e.g., developing or mature cortical neurons. Early studies reported that deficiency of Dicer, the key ribonuclease in miRNA biogenesis, resulted in progressive miRNA loss, death of Purkinje neurons, and cerebellar degeneration [10, 54]. Several neuronal miRNAs have been directly linked to the regulation of key factors involved in AD, including APP and Aβ production and clearance . Although multiple lines of evidence suggest that miRNAs may contribute to the progression of neurodegenerative diseases, the complexity of miRNA regulation in targeting many genes and pathways simultaneously raised concerns about their therapeutic utility as targetable molecules.
One of the most abundant brain-enriched miRNAs is miR-132, which plays a key role in both neuron morphogenesis and plasticity. miR-132, transcribed by the activity-dependent transcription factor CREB, modulates axon and dendrite development and spine maturation in response to a variety of signaling pathways [29, 33]. Deletion of the miR-132 locus decreases dendritic arborization, length, and spine density, impairs integration of newborn neurons, and reduces synapse formation in the adult hippocampus [33, 35, 65]. miR-132 inhibition induces apoptosis in cultured cortical and hippocampal primary neurons via PTEN/AKT/FOXO3 signaling . Notably, miR-132-deficient mice exhibit Tau hyperphosphorylation, aggregation, and decreased memory—all of which are hallmarks of AD [53, 56]. Deletion of miR-132 also fosters Aβ production and plaque accumulation in a triple transgenic mouse AD model . Importantly, multiple studies have shown that miR-132 is the most downregulated miRNA in postmortem AD brain with reductions in miR-132 occurring before neuronal loss and associated with progression of both amyloid and Tau pathology [22, 31, 45, 46, 53, 67].
We hypothesized that supplementation of miR-132 activity may protect against AD and other tauopathies. In support of this idea, we report results of a high-content miRNA screen performed on primary mouse and human neurons treated with either an AD-specific insult (Aβ) or excitotoxic levels of glutamate. Among the miRNAs expressed in the brain, miR-132 exhibits the strongest neuroprotective activity against both Aβ and glutamate. Furthermore, overexpression of miR-132 reduced phosphorylated, acetylated, and cleaved forms of Tau in primary neurons, as well as Tau pathology and caspase-3-dependent apoptosis in PS19 (TauP301S) mice. Functionally, miR-132 overexpression enhanced long-term potentiation (LTP) in WT mice and rescued the impairment of LTP seen in PS19 mice. These results suggest that miR-132 replacement could provide neuroprotection and therapeutic value for Tau-associated neurodegenerative disorders, including AD and FTD.
Materials and methods
Primary neuronal cultures and their analysis
Primary cortical and hippocampal neuron cultures were prepared from WT (E18) and PS19 (P1; JAX:008169) mice, and human fetal cortical specimens (provided by Advanced Bioscience Resources, Alameda, CA, USA). All studies have been approved and performed in accordance with Harvard Medical Area and BWH Standing Committee (IACUC) guidelines. Brain tissues were dissected, dissociated enzymatically by papain, and mechanically by trituration through Pasteur pipette, plated and cultured as previously described . Imaging of the cultures was performed using the IncuCyteTM Live-Cell Imaging System (Essen BioScience). Cell confluency, cell body number, neurite length, and branching points were monitored and quantified using the IncuCyteTM software. Neuron viability was measured using the WST1 assay, following the manufacturer’s instructions (Roche).
Transfections of primary neurons
Transfections of miRNA mimics (Miridian oligonucleotides at 20 nM final concentration, Dharmacon), inhibitors (LNA-containing, at 50 nM, Exiqon), siRNAs (at 25 nM, Dharmacon), and the corresponding control oligonucleotides of the same chemistries to primary mouse and human neurons were carried out using the NeuroMag technology (OZ Biosciences). The cultures were incubated with the transfection mixture in a standard incubator overnight. Half of the media was replaced next morning, and the remaining media were replaced at later time points. Transfection efficacy for miRNA inhibitors, mimics, and siRNA is 95–100% .
SEC isolation of Aβ monomer and preparation of ½-t max Aβ (1–42)
Based on the general consensus that aggregation of Aβ is required for toxicity, we employed a partially aggregated preparation of Aβ(1–42) that contained both amyloid fibrils and Aβ monomer [4, 62]. This preparation is referred to as 1/2tmax, because it is produced by incubating Aβ monomer for a period that yields half of the maximal level of thioflavin T. When used at concentration ≥ 10 μM, 1/2tmax can cause the compromise and death of cultured rodent and human neurons within a period of a few days [4, 62]. Synthetic monomer Aβ (1–42) (human sequence) was obtained from rPeptide (A-1165-2). Briefly, Aβ (1–42) was dissolved at 1 mg/ml in 50 mM Tris–HCl, pH 8.5, containing 7 M guanidinium HCl, and 5 mM ethylenediamine tetraacetic acid, and incubated at room temperature overnight. The sample was then centrifuged at 16,000×g for 30 min and the upper 90% of supernatant applied to a Superdex 75 10/300 size exclusion column (GE Healthcare Biosciences), eluted at 0.5 ml/min with 50 mM ammonium bicarbonate, pH 8.5. Absorbance was monitored at 280 nm. Fractions of 0.5 ml were collected. Peak fractions were pooled and the concentration of Aβ determined using ε275 = 1361/M/cm. Thereafter, ½tmax Aβ(1–42) was prepared as described previously . The samples were aliquoted, flash frozen on dry ice, and stored at − 80 °C.
Real-time quantitative RT-PCR
Total RNA was extracted from cultures and tissues with Exiqon RNA isolation kit, according to the manufacturer’s instructions. For miRNA quantifications, TaqMan® miRNA assays (Life Technologies) were used, and miRNA levels were normalized to the geometrical mean of the uniformly expressed miR-99a, miR-181a, and U6 snRNA. The mRNA levels were monitored by qRT-PCR with specific primers listed in the Supplemental Table S1, on the ViiA-7 System (Thermo Fisher Scientific). Threshold cycles (Cts) were generated automatically, and the relative expressions were shown as 2 −ΔCt . mRNA levels were normalized to the geometrical mean of 18 rRNA, ACTB, and PABP2 mRNAs.
Western blotting analysis
Proteins have been extracted and the concentrations determined by Pierce™ BCA Protein Assay Kit. For Western blot analysis, the proteins have been resolved on the SDS-PAGE, transferred to 0.45 μm nitrocellulose membranes (BioRad), blocked with 5% non-fat dry milk in PBS with 0.1% Tween 20, and processed for immunodetection. Sarkosyl-insoluble tau was isolated as previously described . The following primary antibodies were used following the manufacturer’s instructions: Tau 5, Tau 46, Tau-PHF, Rbfox1, Calpain 2, cleaved Caspase-3, cleaved Caspase-7, GSK3β, EP300, and β-Actin (Cell Signaling). Anti-acetyl-Tau AC312 (rabbit anti-ac-K174 Tau) and MAB359 (rabbit anti-ac-K274 Tau) kindly provided by Li Gan’s laboratory were used at 1/5000 dilution. Antibody detection was performed with the HRP-coupled goat secondary anti-mouse or anti-rabbit antibodies (Immunoresearch), followed by the ECL reaction (Perkin Elmer) and exposure to Fuji X-ray films. The films were scanned and signals quantified using the ImageJ software.
Detection of intracellular and extracellular Tau using ELISAs
Two ELISAs were used in this study. One which is similar to clinically approved assays which employ mid-region directed mAbs and are often erroneously referred to as total tau assays, and the other a novel C-terminal ELISA that uses mAbs specific for the C-terminus and MTBR domains of Tau. The mid-region tau ELISA was performed essentially as described previously . The anti-tau monoclonal antibody BT2 (Thermo Scientific) at 2.5 μg/ml in TBS was used for capture and Tau5 conjugated to alkaline phosphatase was used for detection. Samples were analyzed in duplicate, whereas blanks and Tau441 standards (7.8–8000 pg/ml) were analyzed in triplicate. Standard curves were fitted to a five-parameter logistic function with 1/Y2 weighting, using MasterPlex ReaderFit (MiraiBio). The lower limit of quantification (LLOQ) was calculated for each plate and for the results shown the LLoQ was 31 pg/ml. The C-terminal ELISA was performed exactly as for the mid-region assays except the polyclonal antibody K9JA (243-441aa) was used for capture and the mAb TauAB (425-441aa) was used for detection. For the results shown, the LLoQ of the assay was 7.8 pg/ml.
Cross-linking and immunoprecipitation (iCLIP)
iCLIP was performed according to the published protocol , with minor modifications. Briefly, mouse neurons were irradiated with UV-C light to covalently cross-link proteins to nucleic acids (400 J/m2). Upon cell lysis, samples were subjected to DNase treatment and RNA was partially fragmented using low concentrations of the RNase I (0.002 U/ml, 5 min), followed by the treatment with the RNase inhibitor (RNAsin Plus at 0.5 U/μl) to quench RNase activity. The Rbfox1–RNA complexes were immunopurified using the anti-Rbfox1 antibody (Cell Signaling) immobilized on immunoglobulin G-coated magnetic beads. RNA was isolated and precipitated, and the RT-PCR reactions performed with the Tau-specific primers to amplify different segments of the mRNA.
Validation of miR-132 targets by luciferase reporter assay
Full-length 3′ UTR sequences of Gsk3β, Calpain2, and Rbfox1 were cloned into psiCHECK2 plasmid (Promega, C8021) downstream of renilla luciferase, using XhoI and NotI. Mutations in the miR-132 binding sites were introduced to these constructs using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). Primers used for cloning and mutagenesis are indicated in Table S1. Four hundred nanogram of the constructs were co-transfected with either miRNA mimics (25 nM final concentration) or LNA inhibitors (50 nM), in Lipofectamine 2000, to the SH-SY5Y cells grown in 96-well plates. Alternatively, for primary neurons, 1 µg of the constructs was used per well in 24-well plates. Two days after transfections, luciferase luminescence was measured using the Dual-Glo Luciferase Assay System (Promega, E2920) and detected with Infinite F200 plate reader (TECAN). Renilla luminescence was normalized with that of firefly and the signals were presented as renilla/firefly relative luminescence.
Lentivirus production and stereotaxic brain injections
For lentivirus production, the miR-132-expressing PL13-pSyn-mmu-miR-132-IRES2-EGFP or control PL13-pSyn-IRES2-EGFP plasmid was co-transfected with packaging psPAX2 plasmids and VSV-G envelope-expressing plasmid (Addgene plasmids #12259 and #12260), and the viruses concentrated by additional ultracentrifugation at 25,000 rpm. Lentivirus titers were determined by PCR and functional titer was further determined by serial dilutions in 293T cells, using GFP fluorescence. The titer was estimated using the following formula: titer (TU/ml) = number of transduced cells in day 1 × percentage of GFP+ cells × 1000/volume of lentivirus used (ml). The lentivirus-expressing miR-132 (LV-miR132) or empty vector (EV) (2 μl) was stereotactically injected at 6 × 106 TU/ml to the CA1 region of the right hippocampus (Bregma coordinates: 2.5 mm posterior, 1.7 mm lateral, and 1.8 mm ventral) P–A, 0.5 mm; C–L, 1.7 mm; D–V, 2.3 mm) of C57BL/6J and PS19 mice. The animals were randomized to the treatment and control groups. All animal studies have been approved and performed in accordance with Harvard Medical Area and BWH Standing Committee for Animal Care (IACUC) guidelines.
Mice were sacrificed by CO2 exposure following cervical dislocation, and the brains fixed in 4% paraformaldehyde, embedded, and cryo-sectioned. The 16-μm-thick sections were immunostained for NeuN, GFAP, Cleaved Caspase-3, and Tau-PFH with antibodies from Cell Signaling. The sections were first incubated in the blocking solution (7.5% NGS; 0.4% Triton; 1% BSA; PBS) for 2 h, followed by the overnight incubation in antibody-containing solution (5% NGS; 0.2% Triton; 0.5% BSA; PBS), and 2.5-h incubation with a secondary antibody (either AlexaFluor 568 or AlexaFluor 488; Invitrogen). IHC was visualized by Zeiss confocal microscopy at 20× magnification, and the images were processed with a computerized image analysis system (ZEN 2012 SP2 Software, Zeiss).
Mouse brains were removed and submerged in ice-cold oxygenated cutting solution. Transverse slices (350-μm-thick) were cut with a vibroslicer from the middle portion of each hippocampus. Slices were incubated in artificial cerebrospinal fluid (ACSF), transferred to the recording chamber, and continuously perfused in ACSF saturated with 95% O2 and 5% CO2. Field excitatory postsynaptic potentials (fEPSP) were recorded in the CA1 region of the right hippocampus. Test responses were recorded for 20–30 min before the experiment. LTP was induced by two consecutive trains (1 s) of stimuli at 100 Hz separated by 20 s. The field potentials were amplified using Axon Instruments 200B amplifier and digitized with Digidata 1322A. Traces were obtained by pClamp 9.2 and analyzed using the Clampfit 9.2.
A miRNA screen identifies miR-132 as strongly neuroprotective against Aβ and glutamate excitotoxicity in primary neurons
Overexpression of miR-132 preserves cell body clusters and neurite integrity in WT and PS19 neurons treated with ½ t max Aβ
To investigate the protective effects of miR-132 overexpression in vitro and in vivo, we used a PS19 tau transgenic mouse line which expresses human 1N4R Tau bearing the P301S mutation associated with FTD . PS19 primary neurons were transfected with either anti-miR-132, miR-132 mimic, or control oligonucleotides, and then treated with Aβ. As with WT neurons, in PS19 primary neurons, the miR-132 mimic protected against Aβ, while the anti-miR-132 exacerbated sensitivity to Aβ (Fig. 2d). To investigate the effects of miR-132 overexpression on neuronal morphology in normal versus Aβ-stressed cultures, we imaged live cells over a 5-day interval (Fig. 2e). Of note, naïve PS19 cultures appeared less healthy than WT cultures, exhibited fewer and more clustered cell bodies, and had shorter and less branched neurites. In both WT and PS19 neurons stressed with ½tmax Aβ, miR-132 mimic increased the number of healthy cell bodies, neurite length, and branch points versus neurons transfected with scrambled oligonucleotides (Fig. 2f–h). These data demonstrate that, under stress or toxic conditions, miR-132 rescues neuritic loss and helps to maintain neuronal integrity in both WT and mutant Tau neurons.
miR-132 reduces the levels of total and post-translationally modified forms of Tau, its cleavage, and release in PS19 neurons
Western blot analysis revealed that PS19 primary neurons transfected with miR-132 exhibited slightly reduced levels of total Tau and substantial reduction of Tau phosphorylated at Ser396 and Ser404 (PHF1 epitope) and acetylated at K174 and K274 (Fig. 3b–d). Quantification of three independent experiments indicated that the reduction of post-translationally modified Tau isoforms was more pronounced than that of total Tau (Fig. 3d). These data indicate that the observed decrease in the levels of phosphorylated and acetylated Tau was not merely a consequence of reduced total Tau. Additional analysis of major Tau fragments in PS19 neurons using an antibody against the C-terminal region (Tau46) revealed that miR-132 also reduced the levels of ~ 36 and ~ 17 kDa Tau fragments (Fig. 3e, f), the latter being previously characterized as a potentially neurotoxic fragment(s) produced by Calpain 2 and Caspase 3 proteolytic activities, significant amounts of which were found in the brains of patients with tauopathies [13, 14, 50, 51]. Two distinct sandwich ELISA assays, one based on Tau mid-region detection, reflective of total Tau, and the other based on the detection of C-terminal fragments (capture and detection antibodies are illustrated in Fig. 3a), confirmed that miR-132 produces a small (~ 20%) but a significant reduction of the levels of mid-region-containing Tau, and stronger reduction in the levels of C-terminal-containing Tau (Fig. 3g).
It is now widely appreciated that Tau exists both inside and outside of neurons [5, 28, 47]. Under normal circumstances, the majority of extracellular Tau is C-terminal truncated [28, 38, 61], but it has been speculated that, in disease, the C-terminally truncated forms of Tau are released and may contribute to the seeding and spreading of tau aggregates . Since miR-132 diminishes C-terminal Tau fragments inside primary neurons, we next asked whether it may also affect the release of Tau. Notably, although the concentrations of extracellular mid-region-containing tau were unaffected by miR-132 (Fig. 3h, top), the levels of extracellular C-terminal-containing secreted fragments were strongly reduced (Fig. 3h, bottom). Collectively, these results indicate that Tau homeostasis is regulated by miR-132 at several levels, including the regulation of its post-translational modifications, cleavage, and release from neurons.
miR-132 directly targets the Tau modifiers Rbfox1, GSK3β, EP300, and Calpain 2
Acetyltransferase EP300 is the major acetylase of Tau at K174 implicated in its aggregation and neurodegeneration in AD . It has been previously reported as one of the miR-132 targets contributing to its pro-survival/anti-apoptotic function . Indeed, miR-132 mimics reduced expression of EP300, at both mRNA and protein levels in primary neurons (Fig. 4a, b). These data suggest that the observed miR-132 effects on Tau acetylation (see Fig. 3b, d) could be directly mediated by EP300.
Rbfox1, also known as Ataxin-2-binding protein 1, is an RNA-binding protein (RBP) that plays a pivotal role in alternative splicing, mRNA stability, and translation in the brain [1, 32]. Rbfox1 was predicted as another highly scored miR-132 target; indeed, miR-132 repressed both Rbfox1 mRNA and protein (Fig. 4a, b). Furthermore, the direct binding and reciprocal regulation of Rbfox1 by miR-132 mimic and inhibitor were validated using the luciferase reporters bearing the WT and mutant Rbfox1 3′UTR, as described above for the GSK3β (Fig. 4c, d). We hypothesized that Rbfox1 may regulate Tau mRNA splicing and/or stability. Indeed, silencing of Rbfox1 by RNAi reduced total mRNA and protein levels of Tau in primary neurons (Fig. 4e, f). Using iCLIP approach (Fig. S4a), we determined that Rbfox1 directly binds to Tau mRNA (Fig. 4g), preferentially via the GCAUG motif site found in its coding region (Fig. S4b, c). Therefore, while the exact molecular mechanism remains to be established, Rbfox1 appears as a novel RBP that promotes Tau expression. All together, these data strongly suggest that miR-132 directly regulates Rbfox1 and thereby reduces Tau mRNA stability and/or translation.
Finally, we observed that several proteases implicated in Tau cleavage, including Caspases 3 and 7, and Calpain 2, are regulated by miR-132 (Fig. 4h). One of them, Calpain 2, has been predicted as miR-132 target (Fig. 4c). qRT-PCR analysis and luciferase reporter assays confirmed, respectively, that miR-132 reduced Calpain 2 mRNA expression (Fig. 4a), and this effect was mediated by the direct miR-132 binding to the Calpain 2 3′UTR (Fig. 4d). Therefore, miR-132 regulates Calpain 2 expression and may, thereby, regulate Tau cleavage. Additional rescue experiments on neurons co-transfected with anti-miR-132 and siRNAs cognate to either Rbfox1, GSK3β, EP300, or Calpain 2 demonstrated that these targets, indeed, mediated miR-132 control of tau levels, modifications, and cleavage, respectively (Fig. 4i, j). Overall, these data indicate that miR-132 regulates Tau modifiers, including GSK3β, EP300, Rbfox1, and Calpain 2 that collectively contribute to Tau homeostasis in neurons. In addition, Caspases 3/7-mediated cleavage of Tau may also be regulated by miR-132 via PTEN/AKT/FOXO3A signaling .
Overexpression of miR-132 in PS19 mice reduces caspase-3 activation, Tau hyperphosphorylation, and neuron loss
In an additional set of experiments, PS19 mice were stereotactically injected with Lenti-miR132 a total of three times (i.e., at 3, 4.5, and 6 months, 7 mice per group) before onset of pathology, and analyzed at a time point when pathology is obvious in untreated PS19 mice (i.e., 10 months). In accord with our “treatment” study, overexpression of miR-132 prevented neuronal loss and accumulation of PHF-Tau when administered prior to the emergence of tau pathology (Fig. S8). Thus, dependent on the time of administration, neuronal miR-132 can prevent or halt Tau pathology and neurodegeneration.
Overexpression of miR-132 enhances hippocampal LTP in WT mice and restores it in PS19 mice
Several lines of evidence implicate reduced miR-132 activity in AD and related neurodegenerative conditions. First, from many independent attempts to define miRNAs linked to AD pathology, miR-132 has emerged as the top molecule significantly associated with both plaques and tangles in a variety of disease affected brain areas [21, 22, 24, 45, 53, 64, 67]. miR-132 is downregulated starting at Braak III stage, before neuron loss, and miR-132 reduction is evident in phospho-tau-positive neurons [31, 53]. Furthermore, miR-132 downregulation has been described in other neurodegenerative disorders linked to aggregation and accumulation of misfolded protein Tau, including frontotemporal lobar degeneration and progressive supranuclear palsy [6, 21, 55]. Although miR-132 downregulation in the latter classes of tauopathies still requires validation in larger brain cohorts, the data suggest a possible common mechanism underlying miR-132 dysregulation in both AD and primary tauopathies. Second, miR-132 knockout impairs memory formation and retention in adult mice, induces Tau aggregation, and aggravates both tau and amyloid pathologies in transgenic mouse models [22, 31, 53]. Third, our high-content screen for miRNA modulators of neuroprotection against Aβ and glutamate excitotoxicity performed in this study, identified miR-132 as the top hit (Fig. 1). Finally, miR-132 has important regulatory functions in neuron development, synaptic plasticity, and survival. Of note, additional neuroprotective (e.g., miR-29 and miR-129) and “neurotoxic” (e.g., miR-26b and miR-34a) miRNAs identified in our screen have been previously implicated in the regulation of critical genes and pathways in AD [20, 41, 45]; it will be important to investigate these hits in future studies.
Several targets and signaling pathways may underlie miR-132 neuroprotective functions. Some of them, such as p250GAP, RASA1, and MeCP2, mediate the role of miR-132 in neurite extension, arborization, and synaptogenesis . Other targets, such as PTEN, p300, and FOXO3a, counteract AKT pro-survival signaling; their derepression observed in AD neurons and probably caused by miR-132 downregulation may induce expression of the key apoptotic effectors Bim and Puma, leading to activation of caspases and apoptotic signaling , and also promoting Tau cleavage. Furthermore, recent reports suggest certain direct miR-132 targets implicated in Aβ and Tau metabolism, including the Tau mRNA itself . However, miR-132 does not appear to regulate Tau in human neurons directly . Tau homeostasis is tightly controlled at multiple levels, and we report here that miR-132 regulates key factors affecting tau production, post-translational modifications, and proteolysis. Specifically, miR-132 regulates tau phosphorylation (via direct targeting of GSK3β), acetylation (via a EP300), and cleavage (through calpain 2 and caspases-3/7), and it also reduces Tau mRNA via the direct targeting of the RNA-binding protein, Rbfox1. Tau hyperphosphorylation at PHF1 epitope, largely mediated by GSK3β, affects microtubule dynamics and NFT accumulation, which is considered a hallmark cytopathology in AD and other tauopathies . Since we validated both major tau kinase GSK3β and acetylase EP300 as the direct miR-132 targets (Fig. 4, and ), and additional tau kinase CDK5 is also indirectly repressed by miR-132 via NOS1 signaling , thus, miR-132 emerges as the major regulator of the post-translational modifications of Tau.
Our work also demonstrates that miR-132 regulates Tau cleavage and implicates a newly validated target calpain 2, as well as caspases 3 and 7, in this event. Tau is cleaved by multiple proteolytic enzymes which facilitate its degradation and clearance. However, if allowed to accumulate, some of these fragments become aggregated and/or hyperphosphorylated and neurotoxic [18, 51]. For instance, Tau cleavage by calpain 2 produces a 17 kDa neurotoxic fragment, and significant amounts of these fragments are found in the brains of patients with tauopathy [14, 50]. Mutations in calpain in transgenic flies were shown to prevent Tau toxicity . In addition to the proteolysis of Tau, Calpain also cleaves p35, the principal activator of Cdk5, into p25, which results in the hyperactivation of both Cdk5 and GSK3β, and thereby induces tau hyperphosphorylation [8, 30]. Here, we demonstrate that miR-132 not only regulates Calpain 2 directly, but its levels also inversely correlate with the levels of Calpain 2 mRNA in hundreds of AD brains, suggesting that miR-132 is a primary regulator of Calpain 2 expression in the brain, responsible for its upregulation in AD. In addition, caspase 3/7 activity, modulated by miR-132 indirectly, likely through PTEN/FOXO3/Bim signaling , may also contribute to Tau cleavage and the observed release of tau fragments from neurons (Fig. 3d, e).
We also validated Rbfox1, an RNA-binding protein highly expressed in neuronal tissues, as another direct miR-132 target. By binding to the GCAUG element, Rbfox1 plays a pivotal role in alternative splicing, mRNA stability, and translation [1, 32]. The Rbfox1 knockout mice have a significant increase in neuronal excitability in the dentate gyrus , and a recent study identified a link between Rbfox1 protein and AD . We demonstrate that Rbfox1 binds to and stabilizes neuronal Tau mRNA. Altogether, considering the additional miR-132 target PTBP2 previously implicated in Tau mRNA splicing , these data position miR-132 as the principal regulator of various aspects of Tau homeostasis provide a mechanistic link between the miR-132 downregulation and Tau pathology observed in disease.
Overall, our work supports miR-132 as the master regulator of neuronal health. In addition to its distinct functions in synaptogenesis, neuronal activity, plasticity, memory, and neuronal viability [37, 63], miR-132 regulates Tau metabolism, and its downregulation in AD and other neurodegenerative diseases likely promotes pathogenesis by perturbing multiple signaling pathways. Interestingly, an initial increase in miR-132 levels during early AD Braak stages I–II in the human prefrontal cortex has been described, which contrasts with the decrease seen at more advanced stages of the disease . A similar bi-phasic miR-132 expression pattern has been reported in prion disease , suggesting that miR-132 is part of an initial neuroprotective response. Subsequent downregulation, however, can aggravate the effects of Aβ and Tau toxicities . Of note, miR-132 is regulated by the activity-dependent cAMP-response element-binding (CREB) transcription factor, and its expression pattern in the AD brain mimics that of the brain-derived neurotrophic factor . In addition, alterations in DNA methylation that affect gene expression and perhaps the onset of AD  may play a role in miR-132 downregulation in neurons, as demonstrated for some cancer cells .
Collectively, these data suggest that miR-132 replacement in tauopathies, such as AD, may provide a much-desired neuroprotective effect. Lowering Tau alone with antisense oligonucleotides (ASO) has recently been shown as therapeutically beneficial for tauopathies . Here, we provide a proof-of-principle for miR-132 replacement as a novel neuroprotective strategy to reduce Tau pathology and simultaneously promote nerve growth and regeneration, enhance neuronal survival, and, consequently, improve cognition. miR-132 supplementation protects against strong toxic stimuli even in highly vulnerable and damaged Tau-mutant neurons, in vitro and in vivo. It had preventive effects in young presymptomatic PS19 mice and reduced neuronal loss and Tau pathology even when pathology was already established. The PS19 model exhibits broad brain and spinal cord pathology resulting in severe cognitive, motor, and visual impairments [59, 68]. Since our proof-of-concept study relies on the lentivirus-mediated local unilateral miR-132 supplementation to the CA1 region, it did not allow examination of the effects on global readouts such as neurologic and behavioral phenotypes. To overcome this limitation, broader distribution of miR-132 mimics in the CNS will be required.
Notably, small molecules and other types of inhibitors of major miR-132 targets have entered clinical trials. These include inhibitors of GSK3β, EP300, Calpains, as well as Tau-targeting antibodies and ASO drugs [24, 48, 60, 70]. Remarkably, miR-132 is the natural inhibitor of each of these factors. Therefore, its replacement can provide a multi-hit approach and ensure the benefits of combination therapies. MiR-132 replacement strategies for tauopathies will largely rely on the development of miRNA-mimicking oligonucleotides and technologies for their delivery to the brain and leverage recent advances in the field of oligotherapeutics. Notably, the first “breakthrough” oligonucleotide-based drug for a neurologic disease has recently gained fast FDA approval , and many more are at different stages of clinical development for a wide spectrum of neurodegenerative disorders. AD and other tauopathies have so far proven refractory to small molecules and biological drugs, and miRNA mimics emerge as a new and promising class of therapeutics. Our work validates miR-132 as a first-line candidate for development of such neurotherapies.
We thank Dr. Li Gan for providing antibodies for acetylated Tau, Drs. Andy Billinton and Mike Perkington (MedImmune) for the gift of TauAB antibody, and Jie Shen for valuable advice and equipment access. We thank members and advisers of the Tau Consortium for stimulating discussions. This work was supported by grants from Alzheimer’s Association (NIRG-09-132844) and Tau Consortium/Rainwater foundation.
REF and AMK conceived the project and analyzed the data; REF performed most experiments; SL, ZC, TM, SG, ZW, DTB, RR, AC, and AE assisted with experiments; DJS, KCS, and DMW contributed to data analysis; REF and AMK wrote the manuscript. All authors critically reviewed the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 4.Cantlon A, Frigerio CS, Freir DB, Boland B, Jin M, Walsh DM (2015) The familial British dementia mutation promotes formation of neurotoxic cystine cross-linked Amyloid Bri (ABri) oligomers. J Biol Chem 290:16502–16516. https://doi.org/10.1074/jbc.M115.652263 CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Chai X, Wu S, Murray TK, Kinley R, Cella CV, Sims H et al (2011) Passive immunization with anti-Tau antibodies in two transgenic models: reduction of Tau pathology and delay of disease progression. J Biol Chem 286:34457–34467. https://doi.org/10.1074/jbc.M111.229633 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Chen-Plotkin AS, Unger TL, Gallagher MD, Bill E, Kwong LK, Volpicelli-Daley L et al (2012) TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/212 cluster and affects progranulin pathways. J Neurosci Off J Soc Neurosci 32:11213–11227. https://doi.org/10.1523/JNEUROSCI.0521-12.2012 CrossRefGoogle Scholar
- 16.Florenzano F, Veronica C, Ciasca G, Ciotti MT, Pittaluga A, Olivero G et al (2017) Extracellular truncated tau causes early presynaptic dysfunction associated with Alzheimer’s disease and other tauopathies. Oncotarget 8:64745–64778. https://doi.org/10.18632/oncotarget.17371 CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Hébert SS, Wang W-X, Zhu Q, Nelson PT (2013) A study of small RNAs from cerebral neocortex of pathology-verified Alzheimer’s disease, dementia with lewy bodies, hippocampal sclerosis, frontotemporal lobar dementia, and non-demented human controls. J Alzheimers Dis JAD 35:335–348. https://doi.org/10.3233/JAD-122350 CrossRefPubMedGoogle Scholar
- 22.Hernandez-Rapp J, Rainone S, Goupil C, Dorval V, Smith PY, Saint-Pierre M et al (2016) microRNA-132/212 deficiency enhances Aβ production and senile plaque deposition in Alzheimer’s disease triple transgenic mice. Sci Rep 6:30953. https://doi.org/10.1038/srep30953 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Kanmert D, Cantlon A, Muratore CR, Jin M, O’Malley TT, Lee G et al (2015) C-terminally truncated forms of tau, but not full-length tau or its C-terminal fragments, are released from neurons independently of cell death. J Neurosci Off J Soc Neurosci 35:10851–10865. https://doi.org/10.1523/JNEUROSCI.0387-15.2015 CrossRefGoogle Scholar
- 30.Kurbatskaya K, Phillips EC, Croft CL, Dentoni G, Hughes MM, Wade MA et al (2016) Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain. Acta Neuropathol Commun 4:34. https://doi.org/10.1186/s40478-016-0299-2 CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Majer A, Medina SJ, Niu Y, Abrenica B, Manguiat KJ, Frost KL et al (2012) Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice. PLoS Pathog 8:e1003002. https://doi.org/10.1371/journal.ppat.1003002 CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Minogue AM, Stubbs AK, Frigerio CS, Boland B, Fadeeva JV, Tang J et al (2009) Gamma-secretase processing of APLP1 leads to the production of a p3-like peptide that does not aggregate and is not toxic to neurons. Brain Res 1262:89–99. https://doi.org/10.1016/j.brainres.2009.01.008 CrossRefPubMedGoogle Scholar
- 46.Pichler S, Gu W, Hartl D, Gasparoni G, Leidinger P, Keller A et al (2017) The miRNome of Alzheimer’s disease: consistent downregulation of the miR-132/212 cluster. Neurobiol Aging 50:167.e1–167.e10. https://doi.org/10.1016/j.neurobiolaging.2016.09.019 CrossRefGoogle Scholar
- 48.Rao MV, McBrayer MK, Campbell J, Kumar A, Hashim A, Sershen H et al (2014) Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice. J Neurosci Off J Soc Neurosci 34:9222–9234. https://doi.org/10.1523/JNEUROSCI.1132-14.2014 CrossRefGoogle Scholar
- 59.Takeuchi H, Iba M, Inoue H, Higuchi M, Takao K, Tsukita K et al (2011) P301S mutant human tau transgenic mice manifest early symptoms of human tauopathies with dementia and altered sensorimotor gating. PLoS One 6:e21050. https://doi.org/10.1371/journal.pone.0021050 CrossRefPubMedPubMedCentralGoogle Scholar
- 61.Wagshal D, Sankaranarayanan S, Guss V, Hall T, Berisha F, Lobach I et al (2015) Divergent CSF τ alterations in two common tauopathies: Alzheimer’s disease and progressive supranuclear palsy. J Neurol Neurosurg Psychiatry 86:244–250. https://doi.org/10.1136/jnnp-2014-308004 CrossRefPubMedGoogle Scholar
- 62.Walsh DM, Thulin E, Minogue AM, Gustavsson N, Pang E, Teplow DB, Linse S (2009) A facile method for expression and purification of the Alzheimer’s disease-associated amyloid beta-peptide. FEBS J 276:1266–1281. https://doi.org/10.1111/j.1742-4658.2008.06862.x CrossRefPubMedPubMedCentralGoogle Scholar
- 64.Wang Y, Veremeyko T, Wong AH-K, El Fatimy R, Wei Z, Cai W, Krichevsky AM (2017) Downregulation of miR-132/212 impairs S-nitrosylation balance and induces tau phosphorylation in Alzheimer’s disease. Neurobiol Aging 51:156–166. https://doi.org/10.1016/j.neurobiolaging.2016.12.015 CrossRefPubMedGoogle Scholar
- 70.(2018) Ionis Pharmaceuticals Initiates Clinical Study of IONIS-MAPT Rx in patients with Alzheimer’s disease. Ionis Pharm. Inc., Carlsbad. http://ir.ionispharma.com/news-releases/news-release-details/ionis-pharmaceuticals-initiates-clinical-study-ionis-mapt-rx. Accessed 13 Oct 2017
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