Low Molecular Weight Inhibitors Targeting the RNA-Binding Protein HuR.

The RNA-binding protein human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. This protein has been progressively recognized as a relevant therapeutic target for several pathologies, like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here, we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatic approaches, high-throughput virtual screening, and RNA-protein pulldown assays, we demonstrate that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits a dose-dependent HuR inhibition effect in binding experiments. Importantly, the chemical scaffold is new with respect to the currently known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein.

Intracellular and intercellular transport of RNA organelles in CXG repeat disorders: The strength of weak ties.

RNA is a vital biomolecule, the function of which is tightly spatiotemporally regulated. RNA organelles are biological structures that either membrane-less or surrounded by membrane. They are produced by the all the cells and indulge in vital cellular mechanisms. They include the intracellular RNA granules and the extracellular exosomes. RNA granules play an essential role in intracellular regulation of RNA localization, stability and translation. Aberrant regulation of RNA is connected to disease development. For example, in microsatellite diseases such as CXG repeat expansion disorders, the mutant CXG repeat RNA's localization and function are affected. RNA is not only transported intracellularly but can also be transported between cells via exosomes. The loading of the exosomes is regulated by RNA-protein complexes, and recent studies show that cytosolic RNA granules and exosomes share common content. Intracellular RNA granules and exosome loading may therefore be related. Exosomes can also transfer pathogenic molecules of CXG diseases from cell to cell, thereby driving disease progression. Both intracellular RNA granules and extracellular RNA vesicles may serve as a source for diagnostic and treatment strategies. In therapeutic approaches, pharmaceutical agents may be loaded into exosomes which then transport them to the desired cells/tissues. This is a promising target specific treatment strategy with few side effects. With respect to diagnostics, disease-specific content of exosomes, e.g., RNA-signatures, can serve as attractive biomarker of central nervous system diseases detecting early physiological disturbances, even before symptoms of neurodegeneration appear and irreparable damage to the nervous system occurs. In this review, we summarize the known function of cytoplasmic RNA granules and extracellular vesicles, as well as their role and dysfunction in CXG repeat expansion disorders. We also provide a summary of established protocols for the isolation and characterization of both cytoplasmic and extracellular RNA organelles.

The MID1 Protein: A Promising Therapeutic Target in Huntington's Disease.

Huntington's disease (HD) is caused by an expansion mutation of a CAG repeat in exon 1 of the huntingtin (HTT) gene, that encodes an expanded polyglutamine tract in the HTT protein. HD is characterized by progressive psychiatric and cognitive symptoms associated with a progressive movement disorder. HTT is ubiquitously expressed, but the pathological changes caused by the mutation are most prominent in the central nervous system. Since the mutation was discovered, research has mainly focused on the mutant HTT protein. But what if the polyglutamine protein is not the only cause of the neurotoxicity? Recent studies show that the mutant RNA transcript is also involved in cellular dysfunction. Here we discuss the abnormal interaction of the mutant HTT transcript with a protein complex containing the MID1 protein. MID1 aberrantly binds to CAG repeats and this binding increases with CAG repeat length. Since MID1 is a translation regulator, association of the MID1 complex stimulates translation of mutant HTT mRNA, resulting in an overproduction of polyglutamine protein. Thus, blocking the interaction between MID1 and mutant HTT mRNA is a promising therapeutic approach. Additionally, we show that MID1 expression in the brain of both HD patients and HD mice is aberrantly increased. This finding further supports the concept of blocking the interaction between MID1 and mutant HTT mRNA to counteract mutant HTT translation as a valuable therapeutic strategy. In line, recent studies in which either compounds affecting the assembly of the MID1 complex or molecules targeting HTT RNA, show promising results.

Huntingtin and Its Role in Mechanisms of RNA-Mediated Toxicity.

Huntington's disease (HD) is caused by a CAG-repeat expansion mutation in the Huntingtin (HTT) gene. It is characterized by progressive psychiatric and neurological symptoms in combination with a progressive movement disorder. Despite the ubiquitous expression of HTT, pathological changes occur quite selectively in the central nervous system. Since the discovery of HD more than 150 years ago, a lot of research on molecular mechanisms contributing to neurotoxicity has remained the focal point. While traditionally, the protein encoded by the HTT gene remained the cynosure for researchers and was extensively reviewed elsewhere, several studies in the last few years clearly indicated the contribution of the mutant RNA transcript to cellular dysfunction as well. In this review, we outline recent studies on RNA-mediated molecular mechanisms that are linked to cellular dysfunction in HD models. These mechanisms include mis-splicing, aberrant translation, deregulation of the miRNA machinery, deregulated RNA transport and abnormal regulation of mitochondrial RNA. Furthermore, we summarize recent therapeutical approaches targeting the mutant HTT transcript. While currently available treatments are of a palliative nature only and do not halt the disease progression, recent clinical studies provide hope that these novel RNA-targeting strategies will lead to better therapeutic approaches.

Role and Perspective of Molecular Simulation-Based Investigation of RNA-Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding.

Aberrant RNA-protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA-protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.

FOXO1 controls protein synthesis and transcript abundance of mutant polyglutamine proteins, preventing protein aggregation.

FOXO1, a transcription factor downstream of the insulin/insulin like growth factor axis, has been linked to protein degradation. Elevated expression of FOXO orthologs can also prevent the aggregation of cytosine adenine guanine (CAG)-repeat disease causing polyglutamine (polyQ) proteins but whether FOXO1 targets mutant proteins for degradation is unclear. Here, we show that increased expression of FOXO1 prevents toxic polyQ aggregation in human cells while reducing FOXO1 levels has the opposite effect and accelerates it. Although FOXO1 indeed stimulates autophagy, its effect on polyQ aggregation is independent of autophagy, ubiquitin-proteasome system (UPS) mediated protein degradation and is not due to a change in mutant polyQ protein turnover. Instead, FOXO1 specifically downregulates protein synthesis rates from expanded pathogenic CAG repeat transcripts. FOXO1 orchestrates a change in the composition of proteins that occupy mutant expanded CAG transcripts, including the recruitment of IGF2BP3. This mRNA binding protein enables a FOXO1 driven decrease in pathogenic expanded CAG transcript- and protein levels, thereby reducing the initiation of amyloidogenesis. Our data thus demonstrate that FOXO1 not only preserves protein homeostasis at multiple levels, but also reduces the accumulation of aberrant RNA species that may co-contribute to the toxicity in CAG-repeat diseases.

In vivo targeting of miR-223 in experimental eosinophilic oesophagitis.

OBJECTIVES: Eosinophilic oesophagitis (EoE) is characterised by oesophageal inflammation, fibrosis and dysfunction. Micro (mi)-RNAs interfere with pro-inflammatory and pro-fibrotic transcriptional programs, and miR-223 was upregulated in oesophageal mucosal biopsy specimens from EoE patients. The therapeutic potential of modulating miR-223 expression in vivo has not been determined. We aimed to elucidate the relevance of oesophageal miR-223 expression in an in vivo model of EoE by inhibiting miR-223 tissue expression. METHODS: The expression of miR-223 and the validated miR-223 target insulin-like growth factor receptor 1 (IGF1R) protein was determined in our paediatric cohort of EoE patients. A murine model of Aspergillus fumigatus-induced EoE was employed, and oesophagi were assessed for miR-233, IGF1R, T lymphocyte type 2 (T2) cytokine expression and eosinophil infiltration. Mice were treated with antagomirs targeting miR-223 or resveratrol targeting its upstream regulator Midline-1(MID-1). RESULTS: There was an inverse relationship between an increased expression of miR-223 and a decreased IGF1R protein concentration in biopsy specimens from EoE patients. TNF-related apoptosis-inducing ligand deficiency, MID-1 inhibition and resveratrol treatment suppressed miR-223 expression. Furthermore, inhibition of miR-223 and treatment with resveratrol in the oesophagus resulted in an amelioration of EoE hallmark features including eosinophilic infiltration, oesophageal circumference and a reduction in T2 cytokine expression. CONCLUSION: miR-223 has a key role in the perpetuation of EoE hallmark features downstream of TNF-related apoptosis-inducing ligand and MID-1 in an experimental model. These studies highlight a potentially critical role of miRNA function in EoE aetiology. miR-223 expression in the oesophagus may be therapeutically modulated by resveratrol, providing a potential new therapeutic option to be explored in EoE patients for this increasingly prevalent condition.

Effects of heterochronic, non-myeloablative bone marrow transplantation on age-related behavioural changes in mice.

Experiments using heterochronic parabionts, i.e. young and old animals connected surgically and hence developing a shared circulation, have shown that blood-borne factors, transferred from young to old mice and vice versa, play a role in influencing a range of health outcomes associated with advanced age. Previous work has explored the contributory role of plasma-derived factors in mediating such parabiotic effects, including those on aging-associated neural and behavioural impairments. Here, we wanted to identify possible influences that blood-borne cellular factors may have on age-related behavioural phenotypes. Towards this end, we subjected old BALB/c H-2d mice to repetitive non-myeloablative bone marrow transplants (BMT) from young donor animals and assessed effects on behaviour and cognition. We detected expected age-related alterations in our behavioural assays but did not discern any obvious differences between old BMT mice and old control animals. Our study represents the first to look at possible behavioural and cognitive effects of heterochronic, non-myeloablative BMT. Future work should extend this study by including additional behavioural tests in the analysis, addressing whether beneficial effects of BMT may be detectable on other genetic backgrounds and reconciling our findings with those achieved by myeloablative BMT.

Deregulated Splicing Is a Major Mechanism of RNA-Induced Toxicity in Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD.

Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD), but its role for disease initiation and progression has remained incompletely understood. We here show that the transcription factor Stat3 (signal transducer and activator of transcription 3), a canonical inducer of astrogliosis, is activated in an AD mouse model and human AD Therefore, using a conditional knockout approach, we deleted Stat3 specifically in astrocytes in the APP/PS1 model of AD We found that Stat3-deficient APP/PS1 mice show decreased ß-amyloid levels and plaque burden. Plaque-close microglia displayed a more complex morphology, internalized more ß-amyloid, and upregulated amyloid clearance pathways in Stat3-deficient mice. Moreover, astrocyte-specific Stat3-deficient APP/PS1 mice showed decreased pro-inflammatory cytokine activation and lower dystrophic neurite burden, and were largely protected from cerebral network imbalance. Finally, Stat3 deletion in astrocytes also strongly ameliorated spatial learning and memory decline in APP/PS1 mice. Importantly, these protective effects on network dysfunction and cognition were recapitulated in APP/PS1 mice systemically treated with a preclinical Stat3 inhibitor drug. In summary, our data implicate Stat3-mediated astrogliosis as an important therapeutic target in AD.

The Role of MicroRNAs in Spinocerebellar Ataxia Type 3.

More than 90% of the human genome are transcribed as non-coding RNAs. While it is still under debate if all these non-coding transcripts are functional, there is emerging evidence that RNA has several important functions in addition to coding for proteins. For example, microRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes and human diseases. In spinocerebellar ataxia type 3 (SCA3), a devastating neurodegenerative disease, miRNAs are involved in the disease process at different levels, including the deregulation of components of the general miRNA biogenesis machinery, as well as in the cell type-specific control of the expression of the SCA3 disease protein and other SCA3 disease-relevant proteins. However, it remains difficult to predict whether these changes are a cause or a consequence of the neurodegenerative process in SCA3. Further studies using standardized procedures for the analysis of miRNA expression and larger sample numbers are required to enhance our understanding of the miRNA-mediated processes involved in SCA3 disease and may enable the development of miRNA-based therapeutics. In this review, we summarize the findings of independent studies highlighting both the disease-related and cytoprotective roles of miRNAs that have been implicated so far in the disease process of SCA3.

Upregulation of miR-25 and miR-181 Family Members Correlates with Reduced Expression of ATXN3 in Lymphocytes from SCA3 Patients.

BACKGROUND: Spinocerebellar ataxia type 3 (SCA3), the most common spinocerebellar ataxia, is caused by a polyglutamine (polyQ) expansion in the protein ataxin-3 (ATXN3). Silencing the expression of polyQ-expanded ATXN3 rescues the cellular disease phenotype. OBJECTIVE: This study investigated the differential expression of microRNAs (miRNAs), small noncoding RNAs targeting gene expression, in lymphoblastoid cells (LCs) from SCA3 patients and the capability of identified deregulated miRNAs to target and alter ATXN3 expression. METHODS: MiRNA profiling was performed by microarray hybridization of total RNA from control and SCA3-LCs. The capability of the identified miRNAs and their target sites to suppress ATXN3 expression was analyzed using mutagenesis, reverse transcription PCR, immunoblotting, luciferase reporter assays, mimics and precursors of the identified miRNAs. RESULTS: SCA3-LCs showed significantly decreased expression levels of ATXN3 and a significant upregulation of the ATXN3-3'UTR targeting miRNAs, miR-32 and miR-181c and closely related members of the miR-25 and miR-181 family, respectively. MiR-32 and miR-181c effectively targeted the 3'UTR of ATXN3 and suppressed the expression of ATXN3. CONCLUSIONS: The simultaneous upregulation of closely related miRNAs targeting the 3'UTR of ATXN3 and the significantly reduced ATXN3 expression levels in SCA3-LCs suggests that miR-25 and miR-181 family members cooperatively bind to the 3'UTR to suppress the expression of ATXN3. The findings further suggest that the upregulation of miR-25 and miR-181 family members in SCA3- LCs reflects a cell type-specific, protective mechanism to diminish polyQ-mediated cytotoxic effects. Thus, miRNA mimics of miR-25 and miR-181 family members may prove useful for the treatment of SCA3.

Metformin reverses early cortical network dysfunction and behavior changes in Huntington's disease.

Catching primal functional changes in early, 'very far from disease onset' (VFDO) stages of Huntington's disease is likely to be the key to a successful therapy. Focusing on VFDO stages, we assessed neuronal microcircuits in premanifest Hdh150 knock-in mice. Employing in vivo two-photon Ca2+ imaging, we revealed an early pattern of circuit dysregulation in the visual cortex - one of the first regions affected in premanifest Huntington's disease - characterized by an increase in activity, an enhanced synchronicity and hyperactive neurons. These findings are accompanied by aberrations in animal behavior. We furthermore show that the antidiabetic drug metformin diminishes aberrant Huntingtin protein load and fully restores both early network activity patterns and behavioral aberrations. This network-centered approach reveals a critical window of vulnerability far before clinical manifestation and establishes metformin as a promising candidate for a chronic therapy starting early in premanifest Huntington's disease pathogenesis long before the onset of clinical symptoms.

Upregulation of miR-370 and miR-543 is associated with reduced expression of heat shock protein 40 in spinocerebellar ataxia type 3.

Molecular chaperones are important regulators of protein folding and proteasomal removal of misfolded proteins. In spinocerebellar ataxia type 3 (SCA3), the co-chaperone DnaJ homology subfamily B member 1 (DNAJB1 or heat shock protein 40) is recruited to protein aggregates formed by the disease-causing mutant polyglutamine (polyQ) protein ataxin-3 (ATXN3). Over-expression of DNAJB1 reduces polyQ protein toxicity. Here, we identified two miRNAs, miR-370 and miR-543, that function in posttranscriptional regulation of DNAJB1 expression. MiRNAs are small endogenously produced RNAs controlling mRNA stability and play a role in polyQ disease pathogenesis. In human neuronal cultures derived from SCA3 patient-specific induced pluripotent stem cell (iPSC) lines, miR-370 and miR-543 levels are upregulated, while DNAJB1 expression is concurrently reduced. These findings suggest that downregulation of DNAJB1 by these two miRNAs is an early event that could contribute to SCA3 pathogenesis. Inhibition of these two miRNAs in turn could stabilize DNAJB1 and thereby be beneficial in SCA3 disease.

Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis.

Alzheimer's disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-ß (Aß) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aß peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aß in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.

Reducing Mutant Huntingtin Protein Expression in Living Cells by a Newly Identified RNA CAG Binder.

Expanded CAG trinucleotide repeats in Huntington's disease (HD) are causative for neurotoxicity. The mutant CAG repeat RNA encodes neurotoxic polyglutamine proteins and can lead to a toxic gain of function by aberrantly recruiting RNA-binding proteins. One of these is the MID1 protein, which induces aberrant Huntingtin (HTT) protein translation upon binding. Here we have identified a set of CAG repeat binder candidates by in silico methods. One of those, furamidine, reduces the level of binding of HTT mRNA to MID1 and other target proteins in vitro. Metadynamics calculations, fairly consistent with experimental data measured here, provide hints about the binding mode of the ligand. Importantly, furamidine also decreases the protein level of HTT in a HD cell line model. This shows that small molecules masking RNA-MID1 interactions may be active against mutant HTT protein in living cells.

Pharmacological disruption of the MID1/α4 interaction reduces mutant Huntingtin levels in primary neuronal cultures.

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.