Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation.

Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.

ERRγ agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation.

Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.

Mesenchymal Stem Cell Behavior on Soft Hydrogels with Aligned Surface Topographies.

Human mesenchymal stem cells (HMSCs) are important for cell-based therapies. However, the success of HMSC therapy requires large-scale in vitro expansion of these multipotent cells. The traditional expansion of HMSCs on tissue-culture-treated stiff polystyrene induces significant changes in their shape, multipotency, and secretome, leading to early senescence and subdued paracrine activity. To enhance their therapeutic potential, here, we have developed two-dimensional soft hydrogels with imprinted microscale aligned grooves for use as HMSC culture substrates. We showed that, depending on the dimensions of the topographical features, these substrates led to lower cellular spreading and cytoskeletal tension, maintaining multipotency and osteogenic and adipogenic differentiate potential, while lowering cellular senescence. We also observed a greater capacity of HMSCs to produce anti-inflammatory cytokines after short-term priming on these hydrogel substrates. Overall, these soft hydrogels with unique surface topography have shown great promise as in vitro culture substrates to maximize the therapeutic potential of HMSCs.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.

Temporal and spatial transcriptional fingerprints by antipsychotic or propsychotic drugs in mouse brain.

Various types of antipsychotics have been developed for the treatment of schizophrenia since the accidental discovery of the antipsychotic activity of chlorpromazine. Although all clinically effective antipsychotic agents have common properties to interact with the dopamine D2 receptor (D2R) activation, their precise mechanisms of action remain elusive. Antipsychotics are well known to induce transcriptional changes of immediate early genes (IEGs), raising the possibility that gene expressions play an essential role to improve psychiatric symptoms. Here, we report that while different classes of antipsychotics have complex pharmacological profiles against D2R, they share common transcriptome fingerprint (TFP) profile of IEGs in the murine brain in vivo by quantitative real-time PCR (qPCR). Our data showed that various types of antipsychotics with a profound interaction of D2R including haloperidol (antagonist), olanzapine (antagonist), and aripiprazole (partial agonist) all share common spatial TFPs closely homologous to those of D2R antagonist sulpiride, and elicited greater transcriptional responses in the striatum than in the nucleus accumbens. Meanwhile, D2R agonist quinpirole and propsychotic NMDA antagonists such as MK-801 and phencyclidine (PCP) exhibited the contrasting TFP profiles. Clozapine and propsychotic drug methamphetamine (MAP) displayed peculiar TFPs that reflect their unique pharmacological property. Our results suggest that transcriptional responses are conserved across various types of antipsychotics clinically effective in positive symptoms of schizophrenia and also show that temporal and spatial TFPs may reflect the pharmacological features of the drugs. Thus, we propose that a TFP approach is beneficial to evaluate novel drug candidates for antipsychotic development.

Anatomical transcriptome of G protein-coupled receptors leads to the identification of a novel therapeutic candidate GPR52 for psychiatric disorders.

Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a Cre-lox system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.