What the Gut Tells the Brain-Is There a Link between Microbiota and Huntington's Disease?

The human intestinal microbiota is a diverse and dynamic microenvironment that forms a complex, bi-directional relationship with the host. The microbiome takes part in the digestion of food and the generation of crucial nutrients such as short chain fatty acids (SCFA), but is also impacts the host's metabolism, immune system, and even brain functions. Due to its indispensable role, microbiota has been implicated in both the maintenance of health and the pathogenesis of many diseases. Dysbiosis in the gut microbiota has already been implicated in many neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). However, not much is known about the microbiome composition and its interactions in Huntington's disease (HD). This dominantly heritable, incurable neurodegenerative disease is caused by the expansion of CAG trinucleotide repeats in the huntingtin gene (HTT). As a result, toxic RNA and mutant protein (mHTT), rich in polyglutamine (polyQ), accumulate particularly in the brain, leading to its impaired functions. Interestingly, recent studies indicated that mHTT is also widely expressed in the intestines and could possibly interact with the microbiota, affecting the progression of HD. Several studies have aimed so far to screen the microbiota composition in mouse models of HD and find out whether observed microbiome dysbiosis could affect the functions of the HD brain. This review summarizes ongoing research in the HD field and highlights the essential role of the intestine-brain axis in HD pathogenesis and progression. The review also puts a strong emphasis on indicating microbiome composition as a future target in the urgently needed therapy for this still incurable disease.

Searching for non-canonical functions of aminoacyl-tRNA synthases based on mutations carried by Charcot-Marie-Tooth patients / Poszukiwanie niekanonicznych funkcji syntetaz aminoacylo-tRNA w oparciu o mutacje obecne u pacjentów z choroba Charcot-Marie-Tooth.

Charcot-Marie-Tooth (CMT) is a genetic, incurable neurodegenerative disease which etiology is linked to mutations in almost hundred different genes. The disease affects peripheral nerves which control muscle work and their myelin sheath resulting in progressive muscular dystrophy. The most remarkable genes which mutations are associated with CMT phenotype, are genes encoding aminoacyl-tRNA synthases (aaRS). These proteins are enzymes which common role is to catalyze the reaction of amino acids transfer into tRNA molecules and thereby, to participate in translation of genetic code into the language of proteins. aaRS have been gaining new functions resulting from the mutations acquired in the course of evolution. These functions remain unidentified, despite unraveling the binding partners of aaRS. However, the ongoing molecular studies, which focus on mutations carried by CMT patients and model organisms, bring the researchers closer to unravel the novel functions of aaRS and their potential key role in CMT pathogenesis.

COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/ß-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/ß-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/ß-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. METHODS: Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of ß-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. RESULTS: Wnt/ß-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. CONCLUSIONS: Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Keratin 8-deletion induced colitis predisposes to murine colorectal cancer enforced by the inflammasome and IL-22 pathway.

Keratins (K) are intermediate filament proteins important in protection from cellular stress. K8, K18 and K19 are the main components of keratin filaments in colonic epithelia but their role in intestinal diseases remains ambiguous. A function for keratins in intestinal health is supported by the K8-knock-out (K8(-/-)) mouse which manifests an early chronic ulcerative colitis-like inflammatory bowel disease and epithelial hyperproliferation. We tested whether K8(-/-) mice are more susceptible to colorectal cancer (CRC) compared to K8 wild type (K8(+/+)), and K8 heterozygote (K8(+/-)) mice showing increased proliferation but no inflammation. K8(-/-) mice did not develop CRC spontaneously, but had dramatically increased numbers of tumors in the distal colon in the azoxymethane (AOM) and Apc(Min/+) CRC models while neither K8(+/+) nor K8(+/-) mice were susceptible. Upregulation of IL-22 in combination with a complete loss of its negative regulator IL-22BP, and increased downstream STAT3-signaling in K8(-/-) and K8(-/-)Apc(Min/+) colonic epithelia confirmed that the IL-22 pathway, important in inflammation, proliferation and tissue regeneration, was activated. The nearly total loss of IL-22BP correlated with an activated inflammasome leading to increased cleaved caspase-1, and the putative IL-22BP inhibitor, IL-18, as well as a decrease in ALDH1/2. Ablation of K8 in a colorectal cancer cell line similarly resulted in increased IL-18 and decreased ALDH1/2. K8/K18 co-immunoprecipitated with pro-caspase-1, a component of the inflammasome in the colon, which suggests that keratins modulate inflammasome activity and protect the colon from inflammation and tumorigenesis. The K8-null mouse models also provide novel epithelial-derived robust colon-specific CRC models.

Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice.

Human mutations in keratin 8 (K8) and keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with keratin mutations that associate with liver and other diseases.

Vimentin Suppresses Inflammation and Tumorigenesis in the Mouse Intestine.

Vimentin has been implicated in wound healing, inflammation, and cancer, but its functional contribution to intestinal diseases is poorly understood. To study how vimentin is involved during tissue injury and repair of simple epithelium, we induced colonic epithelial cell damage in the vimentin null (Vim-/-) mouse model. Vim-/- mice challenged with dextran sodium sulfate (DSS) had worse colitis manifestations than wild-type (WT) mice. Vim-/- colons also produced more reactive oxygen and nitrogen species, possibly contributing to the pathogenesis of gut inflammation and tumorigenesis than in WT mice. We subsequently describe that CD11b+ macrophages served as the mainly cellular source of reactive oxygen species (ROS) production via vimentin-ROS-pSTAT3-interleukin-6 inflammatory pathways. Further, we demonstrated that Vim-/- mice did not develop colitis-associated cancer model upon DSS treatment spontaneously but increased tumor numbers and size in the distal colon in the azoxymethane/DSS model comparing with WT mice. Thus, vimentin has a crucial role in protection from colitis induction and tumorigenesis of the colon.

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis.

The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the "stemness" of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives.

Shooting at Moving and Hidden Targets-Tumour Cell Plasticity and the Notch Signalling Pathway in Head and Neck Squamous Cell Carcinomas.

Head and Neck Squamous Cell Carcinoma (HNSCC) is often aggressive, with poor response to current therapies in approximately 40-50% of the patients. Current therapies are restricted to operation and irradiation, often combined with a small number of standard-of-care chemotherapeutic drugs, preferentially for advanced tumour patients. Only very recently, newer targeted therapies have entered the clinics, including Cetuximab, which targets the EGF receptor (EGFR), and several immune checkpoint inhibitors targeting the immune receptor PD-1 and its ligand PD-L1. HNSCC tumour tissues are characterized by a high degree of intra-tumour heterogeneity (ITH), and non-genetic alterations that may affect both non-transformed cells, such as cancer-associated fibroblasts (CAFs), and transformed carcinoma cells. This very high degree of heterogeneity likely contributes to acquired drug resistance, tumour dormancy, relapse, and distant or lymph node metastasis. ITH, in turn, is likely promoted by pronounced tumour cell plasticity, which manifests in highly dynamic and reversible phenomena such as of partial or hybrid forms of epithelial-to-mesenchymal transition (EMT), and enhanced tumour stemness. Stemness and tumour cell plasticity are strongly promoted by Notch signalling, which remains poorly understood especially in HNSCC. Here, we aim to elucidate how Notch signal may act both as a tumour suppressor and proto-oncogenic, probably during different stages of tumour cell initiation and progression. Notch signalling also interacts with numerous other signalling pathways, that may also have a decisive impact on tumour cell plasticity, acquired radio/chemoresistance, and metastatic progression of HNSCC. We outline the current stage of research related to Notch signalling, and how this pathway may be intricately interconnected with other, druggable targets and signalling mechanisms in HNSCC.

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich's Ataxia Patients.

Friedreich's ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.

Progress in understanding Friedreich's ataxia using human induced pluripotent stem cells.

INTRODUCTION: Friedreich's ataxia (FRDA) is an autosomal recessive multisystem disease mainly affecting the peripheral and central nervous systems, and heart. FRDA is caused by a GAA repeat expansion in the first intron of the frataxin (FXN) gene, that leads to reduced expression of FXN mRNA and frataxin protein. Neuronal and cardiac cells are primary targets of frataxin deficiency and generating models via differentiation of induced pluripotent stem cells (iPSCs) into these cell types is essential for progress towards developing therapies for FRDA. AREAS COVERED: This review is focused on modeling FRDA using human iPSCs and various iPSC-differentiated cell types. We emphasized the importance of patient and corrected isogenic cell line pairs to minimize effects caused by biological variability between individuals. EXPERT OPINION: The versatility of iPSC-derived cellular models of FRDA is advantageous for developing new therapeutic strategies, and rigorous testing in such models will be critical for approval of the first treatment for FRDA. Creating a well-characterized and diverse set of iPSC lines, including appropriate isogenic controls, will facilitate achieving this goal. Also, improvement of differentiation protocols, especially towards proprioceptive sensory neurons and organoid generation, is necessary to utilize the full potential of iPSC technology in the drug discovery process.

Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway.

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8-/-) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8-/- colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium.

Simple Epithelial Keratins.

Simple epithelial keratins (SEKs) are the cytoplasmic intermediate filament proteins of single-layered and glandular epithelial cells as found in the liver, pancreas, intestine, and lung. SEKs have broad cytoprotective functions, which are facilitated by dynamic posttranslational modifications and interaction with associated proteins. SEK filaments are composed of obligate heteropolymers of type II (K7, K8) and type I (K18-K20, K23) keratins. The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. Reorganization of the SEK network into aggregates called Mallory-Denk bodies (MDBs) is characteristic for specific liver disorders such as alcoholic and nonalcoholic steatohepatitis. To spur further research on SEKs, we here review the methods and potential caveats of their isolation as well as possibilities to study them in cell culture. The existing transgenic SEK mouse models, their advantages and potential drawbacks are discussed. The tools to induce MDBs, ways of their visualization and quantification, as well as the possibilities to detect SEK variants in humans are summarized.

Keratin 8 absence down-regulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism.

Simple-type epithelial keratins are intermediate filament proteins important for mechanical stability and stress protection. Keratin mutations predispose to human liver disorders, whereas their roles in intestinal diseases are unclear. Absence of keratin 8 (K8) in mice leads to colitis, decreased Na/Cl uptake, protein mistargeting, and longer crypts, suggesting that keratins contribute to intestinal homeostasis. We describe the rate-limiting enzyme of the ketogenic energy metabolism pathway, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), as a major down-regulated protein in the K8-knockout (K8(-/-)) colon. K8 absence leads to decreased quantity and activity of HMGCS2, and the down-regulation is not dependent on the inflammatory state, since HMGCS2 is not decreased in dextran sulfate sodium-induced colitis. Peroxisome proliferator-activated receptor α, a transcriptional activator of HMGCS2, is similarly down-regulated. Ketogenic conditions-starvation or ketogenic diet-increase K8(+/+) HMGCS2, whereas this response is blunted in the K8(-/-) colon. Microbiota-produced short-chain fatty acids (SCFAs), substrates in the colonic ketone body pathway, are increased in stool, which correlates with decreased levels of their main transporter, monocarboxylate transporter 1 (MCT1). Microbial populations, including the main SCFA-butyrate producers in the colon, were not altered in the K8(-/-). In summary, the regulation of the SCFA-MCT1-HMGCS2 axis is disrupted in K8(-/-) colonocytes, suggesting a role for keratins in colonocyte energy metabolism and homeostasis.

Comparative cellular toxicity of hydrophilic and hydrophobic microcystins on Caco-2 cells.

Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.