Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using an Hb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

The ChpR transcriptional regulator of Sinorhizobium meliloti senses 3,5,6-trichloropyridinol, a degradation product of the organophosphate pesticide chlorpyrifos

The global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.

Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

Methylation Motifs in Promoter Sequences May Contribute to the Maintenance of a Conserved m5C Methyltransferase in Helicobacter pylori

DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (m5C) MTase that converts GCGC motifs to Gm5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains.

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids

Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

Novel Synthetic Coumarin-Chalcone Derivative (E)-3-(3-(4-(Dimethylamino)Phenyl)Acryloyl)-4-Hydroxy-2H-Chromen-2-One Activates CREB-Mediated Neuroprotection in Aβ and Tau Cell Models of Alzheimer’s Disease

Abnormal accumulations of misfolded Aβ and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer’s disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aβ and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.