PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines.

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.

Structured Docosahexaenoic Acid (DHA) Enhances Motility and Promotes the Antioxidant Capacity of Aged C. elegans.

The human lifespan has increased over the past century; however, healthspans have not kept up with this trend, especially cognitive health. Among nutrients for brain function maintenance, long-chain omega-3 polyunsaturated fatty acids (ω-3 LCPUFA): DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) must be highlighted, particularly structured forms of EPA and DHA which were developed to improve bioavailability and bioactivity in comparison with conventional ω-3 supplements. This study aims to elucidate the effect of a structured triglyceride form of DHA (DHA-TG) on the healthspan of aged C. elegans. Using a thrashing assay, the nematodes were monitored at 4, 8, and 12 days of adulthood, and DHA-TG improved its motility at every age without affecting lifespan. In addition, the treatment promoted antioxidant capacity by enhancing the activity and expression of SOD (superoxide dismutase) in the nematodes. Lastly, as the effect of DHA-TG was lost in the DAF-16 mutant strain, it might be hypothesized that the effects of DHA need DAF-16/FOXO as an intermediary. In brief, DHA-TG exerted a healthspan-promoting effect resulting in both enhanced physical fitness and increased antioxidant defense in aged C. elegans. For the first time, an improvement in locomotive function in aged wild-type nematodes is described following DHA-TG treatment.

Changes in lipid metabolism driven by steroid signalling modulate proteostasis in C. elegans.

Alzheimer's, Parkinson's and Huntington's diseases can be caused by mutations that enhance protein aggregation, but we still do not know enough about the molecular players of these pathways to develop treatments for these devastating diseases. Here, we screen for mutations that might enhance aggregation in Caenorhabditis elegans, to investigate the mechanisms that protect against dysregulated homeostasis. We report that the stomatin homologue UNC-1 activates neurohormonal signalling from the sulfotransferase SSU-1 in ASJ sensory/endocrine neurons. A putative hormone, produced in ASJ, targets the nuclear receptor NHR-1, which acts cell autonomously in the muscles to modulate polyglutamine repeat (polyQ) aggregation. A second nuclear receptor, DAF-12, functions oppositely to NHR-1 to maintain protein homeostasis. Transcriptomics analyses of unc-1 mutants revealed changes in the expression of genes involved in fat metabolism, suggesting that fat metabolism changes, controlled by neurohormonal signalling, contribute to protein homeostasis. Furthermore, the enzymes involved in the identified signalling pathway are potential targets for treating neurodegenerative diseases caused by disrupted protein homeostasis.

CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2.

A single-nucleotide deletion in the stop codon of the nuclear import receptor transportin-3 (TNPO3), also involved in human immunodeficiency virus type 1 (HIV-1) infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by extending the wild-type protein. Here, we generated a patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNP O 3 mutation. The cell model reproduced critical molecular alterations seen in patients, such as TNP O 3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the TNP O 3 mutation via CRISPR-Cas9 editing caused a significant reversion of the pathological phenotypes in edited cells, including a complete absence of the mutant TNPO3 protein, as detected with a polyclonal antibody specific against the abnormal 15-aa peptide. Transcriptomic analyses found that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9-corrected cells showed that 44% of the alterations were rescued toward normal levels. MicroRNAs (miRNAs) analyses showed that around 50% of miRNAs with impaired expression because of the disease were recovered on the mutation edition. In summary, this work provides proof of concept of the potential of CRISPR-Cas9-mediated gene editing of TNP O 3 as a therapeutic approach and describes critical reagents in LGMDD2 research.

Mn(II) Quinoline Complex (4QMn) Restores Proteostasis and Reduces Toxicity in Experimental Models of Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, of the so-called minority diseases, due to its low prevalence. It is caused by an abnormally long track of glutamines (polyQs) in mutant huntingtin (mHtt), which makes the protein toxic and prone to aggregation. Many pathways of clearance of badly-folded proteins are disrupted in neurons of patients with HD. In this work, we show that one Mn(II) quinone complex (4QMn), designed to work as an artificial superoxide dismutase, is able to activate both the ubiquitin-proteasome system and the autophagy pathway in vitro and in vivo models of HD. Activation of these pathways degrades mHtt and other protein-containing polyQs, which restores proteostasis in these models. Hence, we propose 4QMn as a potential drug to develop a therapy to treat HD.

Spectroscopic, Electrochemical, and Biological Assays of Copper-Binding Molecules for Screening of Different Drugs and Plant Extracts against Neurodegenerative Disorders.

Neurodegenerative disorders, caused by prone-to-aggregation proteins, such as Alzheimer disease or Huntington disease, share other traits such as disrupted homeostasis of essential metal ions, like copper. In this context, in an attempt to identify Cu2+ chelating agents, we study several organic compounds (ethylenediaminetetraacetic acid, phenylenediamine, metformin, salicylate, and trehalose) and organic extracts obtained from Bacopa monnieri L., which has been used in Ayurvedic therapies and presented a broad spectrum of biological properties. For this purpose, UV-visible spectroscopy analysis and electrochemical measurements were performed. Further, biological assays were performed in Caenorhabditis elegans models of polyQ toxicity, in an attempt to obtain better insights on neurodegenerative disorders.

Multiview motion tracking based on a cartesian robot to monitor Caenorhabditis elegans in standard Petri dishes.

Data from manual healthspan assays of the nematode Caenorhabditis elegans (C. elegans) can be complex to quantify. The first attempts to quantify motor performance were done manually, using the so-called thrashing or body bends assay. Some laboratories have automated these approaches using methods that help substantially to quantify these characteristic movements in small well plates. Even so, it is sometimes difficult to find differences in motor behaviour between strains, and/or between treated vs untreated worms. For this reason, we present here a new automated method that increases the resolution flexibility, in order to capture more movement details in large standard Petri dishes, in such way that those movements are less restricted. This method is based on a Cartesian robot, which enables high-resolution images capture in standard Petri dishes. Several cameras mounted strategically on the robot and working with different fields of view, capture the required C. elegans visual information. We have performed a locomotion-based healthspan experiment with several mutant strains, and we have been able to detect statistically significant differences between two strains that show very similar movement patterns.

Neuroprotective Effect of IND1316, an Indole-Based AMPK Activator, in Animal Models of Huntington Disease.

Aggregation of mutant huntingtin, because of an expanded polyglutamine track, underlies the cause of neurodegeneration in Huntington disease (HD). However, it remains unclear how some alterations at the cellular level lead to specific structural changes in HD brains. In this context, the neuroprotective effect of the activation of AMP-activated protein kinase (AMPK) appears to be a determinant factor in several neurodegenerative diseases, including HD. In the present work, we describe a series of indole-derived compounds able to activate AMPK at the cellular level. By using animal models of HD (both worms and mice), we demonstrate the in vivo efficacy of one of these compounds (IND1316), confirming that it can reduce the neuropathological symptoms of this disease. Taken together, in vivo results and in silico studies of druggability, allow us to suggest that IND1316 could be considered as a promising new lead compound for the treatment of HD and other central nervous system diseases in which the activation of AMPK results in neuroprotection.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

Metformin treatment reduces motor and neuropsychiatric phenotypes in the zQ175 mouse model of Huntington disease.

Huntington disease is a neurodegenerative condition for which there is no cure to date. Activation of AMP-activated protein kinase has previously been shown to be beneficial in in vitro and in vivo models of Huntington's disease. Moreover, a recent cross-sectional study demonstrated that treatment with metformin, a well-known activator of this enzyme, is associated with better cognitive scores in patients with this disease. We performed a preclinical study using metformin to treat phenotypes of the zQ175 mouse model of Huntington disease. We evaluated behavior (motor and neuropsychiatric function) and molecular phenotypes (aggregation of mutant huntingtin, levels of brain-derived neurotrophic factor, neuronal inflammation, etc.). We also used two models of polyglutamine toxicity in Caenorhabditis elegans to further explore potential mechanisms of metformin action. Our results provide strong evidence that metformin alleviates motor and neuropsychiatric phenotypes in zQ175 mice. Moreover, metformin intake reduces the number of nuclear aggregates of mutant huntingtin in the striatum. The expression of brain-derived neurotrophic factor, which is reduced in mutant animals, is partially restored in metformin-treated mice, and glial activation in mutant mice is reduced in metformin-treated animals. In addition, using worm models of polyglutamine toxicity, we demonstrate that metformin reduces polyglutamine aggregates and restores neuronal function through mechanisms involving AMP-activated protein kinase and lysosomal function. Our data indicate that metformin alleviates the progression of the disease and further supports AMP-activated protein kinase as a druggable target against Huntington's disease.

CRISPR to fix bad blood: a new tool in basic and clinical hematology.

Advances in genome engineering in the last decade, particularly in the development of programmable nucleases, have made it possible to edit the genomes of most cell types precisely and efficiently. Chief among these advances, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and broad applications in biomedicine. In this review, we focus on the use of CRISPR/Cas9 genome editing in the context of hematologic diseases and appraise the major achievements and challenges in this rapidly moving field to gain a clearer perspective on the potential of this technology to move from the laboratory to the clinic. Accordingly, we discuss data from studies editing hematopoietic cells to understand and model blood diseases, and to develop novel therapies for hematologic malignancies. We provide an overview of the applications of gene editing in experimental, preclinical and clinical hematology including interrogation of gene function, target identification and drug discovery and chimeric antigen receptor T-cell engineering. We also highlight current limitations of CRISPR/Cas9 and the possible strategies to overcome them. Finally, we consider what advances in CRISPR/Cas9 are needed to move the hematology field forward.

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation.

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

USH2A Gene Editing Using the CRISPR System.

Usher syndrome (USH) is a rare autosomal recessive disease and the most common inherited form of combined visual and hearing impairment. Up to 13 genes are associated with this disorder, with USH2A being the most prevalent, due partially to the recurrence rate of the c.2299delG mutation. Excluding hearing aids or cochlear implants for hearing impairment, there are no medical solutions available to treat USH patients. The repair of specific mutations by gene editing is, therefore, an interesting strategy that can be explored using the CRISPR/Cas9 system. In this study, this method of gene editing is used to target the c.2299delG mutation on fibroblasts from an USH patient carrying the mutation in homozygosis. Successful in vitro mutation repair was demonstrated using locus-specific RNA-Cas9 ribonucleoproteins with subsequent homologous recombination repair induced by an engineered template supply. Effects on predicted off-target sites in the CRISPR-treated cells were discarded after a targeted deep-sequencing screen. The proven effectiveness and specificity of these correction tools, applied to the c.2299delG pathogenic variant of USH2A, indicates that the CRISPR system should be considered to further explore a potential treatment of USH.

Metformin intake associates with better cognitive function in patients with Huntington's disease.

Huntington's disease (HD) is an inherited, dominant neurodegenerative disorder caused by an abnormal expansion of CAG triplets in the huntingtin gene (htt). Despite extensive efforts to modify the progression of HD thus far only symptomatic treatment is available. Recent work suggests that treating invertebrate and mice HD models with metformin, a well-known AMPK activator which is used worldwide to treat type 2-diabetes, reduces mutant huntingtin from cells and alleviates many of the phenotypes associated to HD. Herein we report statistical analyses of a sample population of participants in the Enroll-HD database, a world-wide observational study on HD, to assess the effect of metformin intake in HD patients respect to cognitive status using linear models. This cross-sectional study shows for the first time that the use of metformin associates with better cognitive function in HD patients.

AMPK activation protects from neuronal dysfunction and vulnerability across nematode, cellular and mouse models of Huntington's disease.

The adenosine monophosphate activated kinase protein (AMPK) is an evolutionary-conserved protein important for cell survival and organismal longevity through the modulation of energy homeostasis. Several studies suggested that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. However, in Huntington's disease (HD), AMPK may be activated in the striatum of HD mice at a late, post-symptomatic phase of the disease, and high-dose regiments of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide may worsen neuropathological and behavioural phenotypes. Here, we revisited the role of AMPK in HD using models that recapitulate the early features of the disease, including Caenorhabditis elegans neuron dysfunction before cell death and mouse striatal cell vulnerability. Genetic and pharmacological manipulation of aak-2/AMPKα shows that AMPK activation protects C. elegans neurons from the dysfunction induced by human exon-1 huntingtin (Htt) expression, in a daf-16/forkhead box O-dependent manner. Similarly, AMPK activation using genetic manipulation and low-dose metformin treatment protects mouse striatal cells expressing full-length mutant Htt (mHtt), counteracting their vulnerability to stress, with reduction of soluble mHtt levels by metformin and compensation of cytotoxicity by AMPKα1. Furthermore, AMPK protection is active in the mouse brain as delivery of gain-of-function AMPK-γ1 to mouse striata slows down the neurodegenerative effects of mHtt. Collectively, these data highlight the importance of considering the dynamic of HD for assessing the therapeutic potential of stress-response targets in the disease. We postulate that AMPK activation is a compensatory response and valid approach for protecting dysfunctional and vulnerable neurons in HD.

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans.

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5-trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up-regulating IP3 signalling decreases sensitivity. Tissue-specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.

Targeted next generation sequencing for molecular diagnosis of Usher syndrome.

BACKGROUND: Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing. METHODS: A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations. RESULTS: Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements. CONCLUSIONS: Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity.

The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor ß-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished ß-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.

Integration of ß-catenin, sirtuin, and FOXO signaling protects from mutant huntingtin toxicity.

One of the current challenges of neurodegenerative disease research is to determine whether signaling pathways that are essential to cellular homeostasis might contribute to neuronal survival and modulate the pathogenic process in human disease. In Caenorhabditis elegans, sir-2.1/SIRT1 overexpression protects neurons from the early phases of expanded polyglutamine (polyQ) toxicity, and this protection requires the longevity-promoting factor daf-16/FOXO. Here, we show that this neuroprotective effect also requires the DAF-16/FOXO partner bar-1/ß-catenin and putative DAF-16-regulated gene ucp-4, the sole mitochondrial uncoupling protein (UCP) in nematodes. These results fit with a previously proposed mechanism in which the ß-catenin FOXO and SIRT1 proteins may together regulate gene expression and cell survival. Knockdown of ß-catenin enhanced the vulnerability to cell death of mutant-huntingtin striatal cells derived from the HdhQ111 knock-in mice. In addition, this effect was compensated by SIRT1 overexpression and accompanied by the modulation of neuronal UCP expression levels, further highlighting a cross-talk between ß-catenin and SIRT1 in the modulation of mutant polyQ cytoxicity. Taken together, these results suggest that integration of ß-catenin, sirtuin and FOXO signaling protects from the early phases of mutant huntingtin toxicity.

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-ß and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.